From the Department of Biochemistry, University of
Wisconsin, Madison, Wisconsin, the ¶ Department of Biochemistry,
Vilnius University, Ciurlionio 21, Vilnius, Lithuania, and the
** Department of Medical Microbiology and Immunology, University of
Wisconsin-Madison Medical School, Madison, Wisconsin 53706
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ABSTRACT |
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We established an in
vitro assay for the addition of glycosyl-phosphatidylinositol
(GPI) anchors to proteins using procyclic trypanosomes engineered to
express GPI-anchored variant surface glycoprotein (VSG). The assay is
based on the premise that small nucleophiles, such as hydrazine, can
substitute for the GPI moiety and effect displacement of the membrane
anchor of a GPI-anchored protein or pro-protein causing release of the
protein into the aqueous medium. Cell membranes containing
pulse-radiolabeled VSG were incubated with hydrazine, and the VSG
released from the membranes was measured by carbonate extraction,
immunoprecipitation, and SDS-polyacrylamide gel
electrophoresis/fluorography. Release of VSG was time- and
temperature-dependent, was stimulated by hydrazine, and
occurred only for VSG molecules situated in early compartments of the
secretory pathway. No nucleophile-induced VSG release was seen in
membranes prepared from cells expressing a VSG variant with a
conventional transmembrane anchor (i.e. a nonfunctional GPI
signal sequence). Pro-VSG was shown to be a substrate in the reaction
by assaying membranes prepared from cells treated with mannosamine, a
GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine
was used instead of hydrazine, the released VSG could be precipitated
with streptavidin-agarose, indicating that the biotin moiety was
covalently incorporated into the protein. Hydrazine was shown to block
the C terminus of the released VSG hydrazide because the released
material, unlike a truncated form of VSG lacking a GPI signal sequence,
was not susceptible to proteolysis by carboxypeptidases. These results
firmly establish that the released material in our assay is VSG
hydrazide and strengthen the proof that GPI anchoring proceeds via a
transamidation reaction mechanism. The reaction could be inhibited with
sulfhydryl alkylating reagents, suggesting that the transamidase enzyme
contains a functionally important sulfhydryl residue.
Genes encoding glycosylphosphatidylinositol
(GPI)1-anchored proteins
specify two signal sequences in the primary translation product: an
N-terminal signal sequence for targeting the protein to the endoplasmic
reticulum (ER) and a C-terminal GPI-directing signal sequence directing
the attachment of a GPI anchor (1). Both sequences are removed during
processing of the preproprotein to the mature GPI-anchored form, but
cleavage of the N-terminal signal sequence is not strictly necessary
(1, 2). The assembly of GPI-anchored proteins requires translocation of
the nascent polypeptide chain across the ER membrane and replacement of
the C-terminal signal sequence with a preassembled,
ethanolamine-containing GPI moiety attached to the newly exposed
carboxyl group. The reaction is presumed to be catalyzed by an
ER-localized transamidase enzyme (3, 4).
GPI anchoring can be reproduced in cell-free systems that rely on
endogenous, membrane protein acceptors for GPI anchors (3) or,
alternatively, that involve an in vitro translation system to load microsomal membranes with pro-protein substrates for GPI modification (5, 6). Using such systems, strong, albeit circumstantial
evidence was obtained for a transamidation reaction mechanism. In an
early report, Mayor et al. (3) exploited a trypanosome
cell-free system that had been previously used extensively for studies
of GPI biosynthesis (7, 8) to show that transfer of in situ
synthesized or exogenously supplied radiolabeled GPIs to endogenous
protein acceptors did not require ATP or GTP. The apparent lack of an
energy requirement for GPI anchoring in this system was the first
experimental evidence consistent with a transamidation reaction
mechanism. Although this result is qualitatively useful, quantitatively
only a low level of GPI anchor addition was observed, and the
possibility that a preformed energy source or a preactivated GPI
anchoring enzyme existed in the lysate could not be ruled out.
Other studies pioneered by Udenfriend and co-workers (1, 6) employed a
cell-free system consisting of mammalian cell microsomes capable of
processing in vitro synthesized preproproteins to a
GPI-anchored form. Kodukula et al. (9) developed a
convenient reporter protein for this purpose based on the sequence of
placental alkaline phosphatase (PLAP), a GPI-anchored protein. The
reporter, prepromini-PLAP was found to be sequentially processed by
microsomal enzymes to promini-PLAP (lacking the N-terminal signal
sequence) and GPI-anchored mini-PLAP. The transition from promini-PLAP
to mini-PLAP was found to require ATP, GTP, and ER lumenal proteins, consistent with a chaperone-mediated maturation step prior to GPI
anchoring (10-12). The less stringent GTP requirement for this process
remains enigmatic. However, there appeared to be no requirement for
energy in the final conversion from promini-PLAP to mini-PLAP, consistent with a transamidation mechanism for GPI anchoring.
The main product of the reaction in the mammalian
translation-translocation system described above is GPI-anchored
mini-PLAP. However, a small amount of free mini-PLAP (lacking the
C-terminal signal sequence as well as the GPI anchor) is invariably
formed, probably via a nucleophilic attack by water on the active
carbonyl formed as a result of the initial step of the transamidation
reaction sequence (13). Maxwell et al. (4) developed this
observation further and demonstrated that other nucleophiles such as
hydrazine and hydroxylamine could effectively compete with GPI to
generate a molecule similar to free mini-PLAP. They presumed that the
free mini-PLAP variant generated under these conditions was mini-PLAP hydrazide or mini-PLAP hydroxamate; however, they were unable to
demonstrate the nature of these products because of the subpicomole amounts of material generated in their in vitro
translation-translocation experiments (4). These data, although
incomplete, provide the best published evidence thus far that GPI
anchoring proceeds via a transamidation reaction mechanism.
In this paper we describe a convenient assay for GPI anchoring based on
a cell-free system from insect stage African trypanosomes engineered to
express a well characterized GPI-anchored protein. We use the assay to
demonstrate explicitly that the enzyme-mediated, hydrazine-induced
cleavage of the C-terminal GPI signal sequence from a pro-protein
occurs in early compartments of the secretory pathway, requires a
functional GPI signal sequence, and results in the formation of a
soluble protein product that is modified by hydrazine at its C
terminus. We also demonstrate that sulfhydryl alkylating reagents block
activity, consistent with a role for a free sulfhydryl residue in
catalysis. These data provide proof that GPI anchoring proceeds via a
transamidation reaction mechanism.
Materials--
Protein A-Sepharose was from Amersham Pharmacia
Biotech, biotin-LC-hydrazide, and sulfo-NHS-biotin were from Pierce,
carboxypeptidases P and W were from Calbiochem-Behring Corp. (San
Diego, CA), materials for SDS-polyacrylamide gel electrophoresis were
from Bio-Rad, 10,000 NMWL Eppendorf filter units were from Millipore
Corporation (Bedford, MA), cell culture media were from Life
Technologies, Inc. and Specialty Media Inc.,
[3H]ethanolamine (~30 Ci/mmol) was from American
Radiolabeled Chemicals (St. Louis, MO), and
EXPRE35S35S cysteine/methionine protein
labeling mix (>1,000 Ci/mmol) was from NEN Life Science Products.
Autofluor was from National Diagnostics (Atlanta, GA). All other
reagents were obtained from Sigma.
Growth and Metabolic Labeling of Trypanosomes--
The growth
and maintenance of procyclic trypanosomes, and the generation of stably
transformed procyclic cell lines expressing full-length GPI-anchored
variant surface glycoprotein 117 (VSG 117 or 117wt) and a truncated
form lacking the C-terminal GPI signal sequence (117 Metabolic Radiolabeling of Trypanosomes in the Presence of
Mannosamine--
Procyclic cells were labeled with
[3H]ethanolamine or [35S]Cys/Met in the
absence or presence of mannosamine to obtain GPI-anchored VSG or
non-GPI-anchored VSG proprotein, respectively.
[3H]Ethanolamine labeling of procyclics was carried out
as described previously (17). For [35S]Cys/Met labeling,
procyclics were cultured in glucose-free RPMI 1640 (supplemented with
10% dialyzed fetal calf serum, 10 mM glycerol, 5.5 mM proline, and 33 mM Hepes, pH 7.4) for 30 min. The cultures were then supplemented with 5 mM
mannosamine or water and incubated for a further 90 min. The procyclics
were then resuspended in cysteine-, methionine-, and glucose-free RPMI
1640 (supplemented as above) and labeled as described above.
Preparation of the Trypanosome Cell-free System--
Trypanosome
membranes (trypanosome cell-free system) were prepared from
metabolically radiolabeled cells as described previously (7) except
that the cells were not preincubated with tunicamycin prior to lysis.
Aliquots of membranes (5 × 108 cell equivalents/ml)
were snap-frozen in liquid nitrogen and stored at Trypanosome Cell-free System Incubations and Transamidation
Assay--
The trypanosome cell-free system was used as the enzyme
source. Trypanosome membranes were washed twice in 0.1 M
Hepes buffer, pH 7.5, containing 25 mM KCl, 5 mM MgCl2, 0.1 mM tosyl-lysine chloro-methyl ketone and 2 µg/ml leupeptin and then suspended at
109 cell equivalents/ml in 200 mM Hepes, pH
7.5. Aliquots (25 µl) of this lysate were added to tubes containing
25 µl of 20 mM hydrazine (5 mM hydrazine for
some experiments), 20 mM Hepes, pH 7.5, or 20 mM Hepes, pH 7.5, only. Protease inhibitors were also added to this solution when they were used. The tubes were incubated at
37 °C (except for the experiment shown in Fig. 1B where
temperature dependence of the reaction was investigated) for 45 min.
For incubations with biotin-LC-hydrazide, the hydrazide was dissolved
in Me2SO (50 mM) and 5 µl was added to the
incubation. Corresponding controls where just Me2SO was
added were also carried out. These incubations were for 3 h to
maximize the nucleophile-induced release of VSG. The reactions were
terminated by the addition of 350 µl of 0.1 M sodium
carbonate and placed on ice for 30 min. Sodium carbonate extraction
allowed for the isolation of membrane bound components from those that
are soluble. The solution was then layered on top of a 0.1 M sodium carbonate, 0.5 M sucrose cushion and
spun in a Beckman TLA 100.2 rotor at 90,000 rpm for 30 min. 200 µl of
the resulting supernatant was transferred to a tube containing 26.2 µl of 1.3 M NaCl, 0.9% SDS, 4.35% deoxycholate, 4.35%
Nonidet P-40. The remaining membrane pellet was resuspended in 500 µl of TEN buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, and 5 mM EDTA) containing 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS (TEN-detergent or TEN-D). The samples were then
subjected to immunoprecipitation as described below.
Immunoprecipitation and Electrophoresis--
Affinity purified
rabbit anti-VSG 117 and rabbit anti-VSG 117 antisera were used in these
experiments. Antibody was preadsorbed to protein A-Sepharose (PAS)
beads (14) washed once in TEN-D and resuspended to the original volume.
Typically 50 µl of PAS:antibody suspension was added to 200 µl of
the sodium carbonate washed pellet or supernatant, which was
resuspended in TEN-D. Samples were mixed for 2 h at 4 °C and
then washed three times with TEN-D and once with TEN. For most of the
experiments 25 µl of 2× sample buffer was added to the 25 µl of
PAS beads, and the sample was boiled for 10 min. The tubes were then
spun in a microcentrifuge, and 22 µl of the supernatant was
fractionated by 10% SDS-PAGE (18). Dried gels were analyzed by fluorography.
Treatment of Purified Membrane Form VSG with Sulfo-NHS-biotin and
Biotin-LC-hydrazide--
[3H]Myristic acid-labeled,
GPI-anchored VSG 117 was purified from metabolically labeled
bloodstream stage trypanosomes as described previously (19). This
material was incubated on ice in 100 mM Hepes, pH 8, for 45 min with either sulfo-NHS-biotin (an amine-reactive compound) or
biotin-LC-hydrazide. The mixture was then passed through a prewashed
Millipore ultrafree-MC 10,000 NMWL filter (molecular mass cut-off,
10,000 kDa) to wash away any residual biotin-derivatives
(biotin-LC-hydrazide or sulfo-NHS-biotin) and precipitated with
streptavidin beads or anti-VSG 117 antibody as described below.
Precipitation Using Streptavidin Beads--
In experiments using
biotin derivatives, VSG was analyzed by precipitation with
streptavidin-agarose following immunoprecipitation with anti-VSG
antibodies. The sodium carbonate extract was passed through a prewashed
Millipore ultrafree-MC 10,000 NMWL filter (molecular mass cut-off,
10,000) to wash away any residual biotin-derivatives (biotin-LC-hydrazide or sulfo-NHS-biotin). The VSG captured on the
filter was recovered by washing the filter twice with 400 µl of TEN
buffer and then immunoprecipitating as above. The immunoprecipitated VSG was resuspended with 120 µl of Hepes, pH 7.5, and placed in a
100 °C heating block for 10 min, and the boiled sample was then spun
at 14,000 rpm in a microcentrifuge to remove the antibody-coated beads.
The supernatant (approximately 100 µl containing immunopurified VSG)
was then supplemented with 100 µl of 4× TEN-D buffer and 200 µl of
water. Streptavidin coupled to 6% agarose beads was washed twice with
TEN-D buffer and resuspended (also in TEN-D) to its original volume. 50 µl of this was added to the immunopurified VSG (in TEN-D) and mixed
for 9 h at 4 °C. The samples were then washed three times with
TEN-D and once with TEN. Subsequently, 25 µl of 2× sample buffer was
added to the streptavidin beads, and the sample was boiled for 15 min
and analyzed by SDS-PAGE and fluorography or taken directly for liquid
scintillation counting. Precipitation efficiency and sample recovery
using streptavidin-agarose was ~4.5% versus ~90% for
precipitation with anti-VSG 117 antibodies.
Analysis of the Site of Hydrazide Incorporation into VSG
Hydrazide--
Metabolically radiolabeled VSG hydrazide was analyzed
by carboxypeptidase treatment and SDS-PAGE/fluorography as follows. [35S]Cys/Met-labeled VSG hydrazide was generated as
described and immunoprecipitated using anti-VSG antibodies preadsorbed
to PAS beads. The beads were repeatedly washed with 0.1 M
glycine, pH 2.5, to release the bound material. The immunoisolated VSG
hydrazide was dialyzed against water (using a dialysis membrane with a
12,000-14,000-Da molecular mass cut-off), dried in a centrifugal
evaporator, resuspended in water, boiled, cooled, and then made up to
50 mM NaOAc, pH 4.5. The sample was then incubated with a
mixture of carboxypeptidases P and W (140 milliunits and 30 units,
respectively) for 18 h at 30 °C. At the end of the incubation
period, SDS-PAGE sample buffer was added, and the sample was analyzed
by SDS-PAGE/fluorography. Radiolabeled 117 Estimate of the Amount of VSG Hydrazide Generated per
Assay--
Mayor et al. (3) estimated that lysates from
bloodstream stage trypanosomes contain approximately 400-4,000 VSG
molecules per cell equivalent that could act as acceptors in the
transamidase reaction; the procyclic lysates used in our experiments
have ~5% of the number of acceptors reported for bloodstage cells
(22), i.e. 20-200 molecules/cell equivalent. Under standard
assay conditions using 25 µl of lysate (2.5 × 107
cell equivalents), this implies release of 5 × 108 to
5 × 109 VSG hydrazide molecules, i.e.
0.8-8 fmol (0.05-0.5 ng of protein).
High Performance TLC Analysis--
Lipids were extracted from
radiolabeled trypanosomes as described previously (Ref. 20; see also
legend to Fig. 3), and analyzed by TLC using 10-cm glass-backed silica
gel 60 high performance TLC plates (Merck) and chloroform/methanol/1
M ammonium acetate/13 M ammonium
hydroxide/water (180:140:9:9:23, v/v/v/v/v) as the solvent system.
Radiolabeled components in the chromatogram were visualized using a
Berthold LB2842 linear scanner.
GPI-specific Phospholipase D Analysis of
[3H]Ethanolamine-labeled Lipid Extract--
GPI-specific
phospholipase D treatment was performed as described (21), using human
serum as the enzyme source.
GPI Anchoring in a Mammalian Cell-free
System--
Prepromini-PLAP mRNA (prepared from plasmid
pGEM-4Z/mini-PLAP A Cell-free Assay for GPI Anchoring: VSG Is Released from
Trypanosome Lysates in a Time- and Temperature-dependent
Manner in the Presence of Nucleophiles--
We describe a cell-free
assay for GPI anchoring based on the premise that the putative GPI
transamidase (GPIT) can use small nucleophiles such as hydrazine to
effect the displacement of the GPI signal sequence or GPI anchor from a
GPI proprotein or GPI-anchored protein, respectively, resulting in the
release of a water-soluble derivative of the protein (4). To
demonstrate the principle of the assay, we used lysates prepared from
procyclic trypanosomes engineered to express VSG 117 (14), a
conveniently detectable GPI-anchored protein. The cells were
metabolically radiolabeled with [35S]cysteine/methionine
for 15 min before being washed, osmotically lysed, and washed again.
When the labeled crude membranes (containing membrane-associated,
radiolabeled VSG) were incubated with hydrazine and then processed
by carbonate extraction to separate water-soluble molecules from
membrane-associated material, VSG was detected in the carbonate
extract. Analysis of the released material by immunoprecipitation with
anti-VSG antibodies, SDS-PAGE, and fluorography confirmed that the
released material was a ~58-kDa VSG molecule, comparable in size to
an anchorless VSG variant expressed in procyclic trypanosomes (14)
(also see Fig. 6). Production of the released material was dependent on
hydrazine, time (Fig. 1A,
left panel), and temperature (Fig. 1B). As we
show later (see Fig. 5), the released material is a hydrazide
derivative of VSG, lacking a membrane anchor. VSG release was also
observed when the labeled membranes were incubated with hydroxylamine
instead of hydrazine (Fig. 1A, right panel,
left-hand lane). These results suggest that the production
of a soluble derivative of VSG from a membrane-bound precursor in the
presence of nucleophiles is due to temperature-dependent enzymatic activity.
Additional experiments were performed to confirm that
nucleophile-induced release of VSG shared some of the characteristics of the GPI anchoring reaction. We specifically set out to show that
nucleophile-induced VSG release depended on (i) the cellular location
of the VSG molecule and (ii) the presence of either a GPI signal
sequence or a GPI anchor at the C terminus of VSG.
The Enzyme Responsible for Nucleophile-induced VSG Release Is
Located Early in the Secretory Pathway--
A pulse-chase analysis
revealed that membranes prepared from cells labeled with
[35S]cysteine/methionine for 15 min and then chased for
1 h with complete medium showed very little hydrazine-induced
release of VSG when compared with lysates from 15 min pulse-labeled
cells (Fig. 2A, compare
lanes 1 and 2 with lanes 3 and
4). Samples were prepared from cells metabolically labeled
in the presence of 3 mM 1,10-phenanthroline to block the
activity of a surface metalloprotease that cleaves VSG molecules when
they arrive at the cell surface (14). Based on the half-time (~1.2 h)
for export of GPI-anchored VSG to the plasma membrane (22), we would
expect that the pulse-labeled samples contain a significant proportion
of radiolabeled VSG in the ER, whereas the amount of ER-localized VSG
should be greatly reduced in the chase samples (14). We conclude that
the hydrazine-induced release of VSG is specific for molecules
located in early compartments of the secretory pathway such as the ER,
consistent with a wide variety of data indicating that GPI anchoring is
an ER-localized event (11, 23).
VSG 117tm, a VSG Derivative Anchored by a Transmembrane Domain,
Cannot Be Enzymatically Processed in the Presence of Hydrazine to Yield
a Soluble Product--
In a separate experiment to characterize
nucleophile-induced VSG release, procyclic trypanosomes expressing a
transmembrane form of VSG 117 (117tm, a VSG molecule with its
C-terminal GPI addition sequence replaced with the transmembrane region
of p67, a lysosomal protein in T. brucei (16)) were
pulse-labeled, and lysates were prepared. On incubation of these
lysates with hydrazine, no VSG release was seen (Fig. 2B).
Thus a VSG variant with a nonfunctional C-terminal GPI signal sequence
cannot act as a substrate in the assay. This result suggests that
hydrazine-stimulated VSG release requires VSG substrates with either a
GPI signal sequence or a GPI anchor. This observation is consistent
with the idea that the activity being observed in this assay is the one
that usually adds GPI anchors onto proteins.
VSG Pro-protein Can Be Enzymatically Processed in the Presence of
Hydrazine to Yield a Soluble Product--
The pulse-labeled lysates
used in the experiments described above contain predominantly
GPI-anchored VSG. However, they also likely contain small amounts of
unprocessed VSG pro-protein bearing a GPI signal sequence. Both these
forms of the protein are potential substrates for the GPI transamidase.
To establish whether nucleophile-induced VSG release could originate
directly from enzymatic processing of the VSG pro-protein, we
established conditions where GPI biosynthesis and GPI anchoring of
proteins were abolished. Under these conditions, the only radiolabeled
VSG molecules present in the lysates would be pro-forms possessing a
GPI signal sequence. To generate a GPI biosynthesis defect we incubated
the cells with mannosamine, a compound that when metabolized blocks GPI
synthesis prior to the addition of the third mannose residue and
phosphoethanolamine cap (17, 24).
Lipid extracts from procyclic trypanosomes metabolically radiolabeled
with [3H]ethanolamine contain the mature GPI structure
PP1
(ethanolamine-PO4-Man
To assess the effect of mannosamine on
[3H]ethanolamine labeling of proteins,
[3H]ethanolamine-labeled cells were analyzed for
[3H]ethanolamine-labeled VSG by immunoprecipitation,
SDS-PAGE, and fluorography. The fluorogram was quantitated by
densitometry. As shown in Fig. 3B,
[3H]ethanolamine labeling of VSG was almost completely
abolished in mannosamine-treated cells, similar to results obtained by
others (17).
The above controls establish that GPI synthesis and the production of
GPI-anchored proteins are essentially abolished in mannosamine-treated procyclic trypanosomes, indicating that mannosamine treatment prior to
and during pulse-labeling with [35S]cysteine/methionine
would yield cell membranes containing radiolabeled pro-VSG and no
GPI-anchored VSG. When membranes prepared from mannosamine-treated
cells were incubated with hydrazine, VSG release was detected similar
to that seen with control membranes from untreated cells (Fig.
3C, lanes 3 and 4; control samples are
shown in lanes 1 and 2). This result indicates
that the VSG pro-protein can be directly processed by the GPI
transamidase in the presence of hydrazine to yield a soluble product.
Sulfhydryl Alkylating Reagents Inhibit Nucleophile-induced VSG
Release--
It has previously been shown that the GPI anchoring
reaction in bloodstream stage trypanosomes is inhibited by the
sulfhydryl alkylating reagent p-chloromercuriphenyl sulfonic
acid (pCMPSA) (3). We tested the effect of pCMPSA as well as two other
reagents (iodoacetamide and p-chloromercuribenzoate (pCMB))
on nucleophile-induced VSG release. All three compounds inhibited
hydrazine-induced release of VSG (Fig.
4A, compare lane 3 (no inhibitor) with lanes 4 (pCMPSA) and 5 (iodoacetamide)) and compare lane 7 (no inhibitor) with lane 6 (pCMB)). This result is consistent with the proposal
that the trypanosome GPI transamidase contains a catalytically
important sulfhydryl residue. Another possible but less likely
interpretation is that alkylation of a sulfhydryl residue(s) elsewhere
in the protein results in inhibition of transamidase activity.
The same inhibitors were tested in a mammalian cell-free system to see
if they caused inhibition of the mammalian GPI anchor addition
reaction. Messenger RNA corresponding to prepromini-PLAP, a model
protein with an N-terminal signal sequence and a C-terminal GPI
addition sequence (9), was translated in the presence of thymoma cell
microsomes for 30 min at 27 °C, and the incubation was continued for
an additional 90 min in the presence or absence of hydrazine and the
various sulfhydryl alkylating reagents. The sulfhydryl alkylating
reagents could not be introduced at the outset because they inhibit
protein translocation (27). The samples were then processed by
proteinase K treatment to eliminate nontranslocated prepromini-PLAP and
analyzed by SDS-PAGE and fluorography. The results of the experiment
are shown in Fig. 4B. In the absence of inhibitors and
hydrazine, two products are seen: promini-PLAP and GPI-anchored
mini-PLAP (lane 1). When hydrazine was included in the
90-min incubation, a lower molecular mass product was formed (lane 2) (4, 13). The pattern of bands, particularly the ratio of mini-PLAP-hydrazine to mini-PLAP, seen in lanes 1 and 2 of Fig. 4B was unaltered in the presence of
pCMPSA (lanes 3 and 4), pCMB (lanes 5 and 6), and iodoacetamide (lanes 7 and
8), indicating that the sulfyhydryl alkylating reagents do
not affect GPI anchoring in mammalian cells. The lack of effect by
pCMPSA may be due to its inability to cross microsomal membranes, but the analogous reagent pCMB, as well as iodoacetamide, are
membrane permeant and would be expected to react with lumenal targets
(40). Although these results appear to suggest that mammalian GPIT has a different inhibition profile from the trypanosome GPIT, an
alternative explanation may have to do with the way in which the assay
was set-up. Because sulfhydryl alkylating reagents cannot be added at
the outset of the assay because of their inhibitory effect on protein
translocation (27), it is possible that most of the GPIT is complexed
to mini-PLAP (e.g. through a thioester bond between a
sulfhydryl residue in GPIT and the Hydrazine Is Covalently Incorporated into VSG as a Consequence of
GPIT Action--
A transamidation reaction mechanism predicts that in
our assay format an amine containing nucleophile (H2N-X)
becomes covalently attached to the reaction substrate, i.e.
the VSG pro-protein (or GPI-anchored VSG), with concomitant
displacement of the GPI signal sequence (or GPI anchor) (see Fig. 7).
To test this we used a modified hydrazine molecule,
biotin-LC-hydrazide, containing a biotin moiety amide-linked by a
spacer arm to one of the nitrogen atoms in hydrazine (see Fig. 7).
Incubation of procyclic membranes with biotin-LC-hydrazide resulted in
VSG release (Fig. 5A, compare
lanes 3 and 4), although the efficiency of
release was somewhat lower than that seen with hydrazine (Fig.
5B, lanes 1 and 2). The VSG released
from the membranes by incubation with either hydrazine or
biotin-LC-hydrazide was immunoprecipitated with anti-VSG antibodies,
released from the antibody beads by boiling, separated from the beads
by centrifugation, and then precipitated with streptavidin-agarose.
Control samples using only an anti-VSG 117 immunoprecipitation step
showed the normal profile of VSG release from membranes with both
hydrazine and the biotin-LC-hydrazide (Fig. 5A, lanes
1-4). Identically treated samples (containing eight times as much
material to compensate for the inefficiency of precipitation and sample
recovery using streptavidin beads; see "Experimental Procedures")
subjected to the additional streptavidin precipitation step showed a
radioactive band corresponding to VSG 117 only in the sample treated
with biotin-LC-hydrazide (Fig. 5A, lane 8); no
VSG was recovered in the streptavidin precipitate of the sample treated
with hydrazine (Fig. 5A, lane 5), indicating the
specificity of the streptavidin precipitation analysis.
We considered the possibility that biotin-LC-hydrazide was reacting
with VSG nonenzymatically, resulting in the formation of the
biotinylated VSG molecule that we detected by streptavidin precipitation. To address this issue and further verify the specificity of the streptavidin precipitation analysis, we analyzed a sample of
purified [3H]myristic acid-labeled, GPI-anchored VSG 117 incubated with either biotin-LC-hydrazide or sulfo-NHS-biotin (an
amine-reactive biotin derivative expected to modify free amines on VSG)
in the absence of membranes. After incubation, the samples
were washed on low molecular mass cut-off filters to remove unreacted
biotin reagents, and the VSG molecules, captured on the filters, were
resuspended and treated with streptavidin agarose. VSG exposed to
sulfo-NHS-biotin was precipitated with streptavidin, whereas VSG
treated with biotin-LC-hydrazide did not bind streptavidin (Fig.
5B). These results, together with the temperature dependence
profile (Fig. 1B), strongly indicate that under our usual
assay conditions the VSG released from the membranes in the presence of
biotin-LC-hydrazide (Fig. 5A, lanes 4 and
8) is an enzymatically generated hydrazide derivative and contains covalently incorporated biotin.
Hydrazine Is Incorporated at or near the C Terminus of
GPIT-released VSG Hydrazide--
In preliminary attempts to locate the
site of hydrazine modification in VSG hydrazide by mass spectrometry we
concluded that the amount of starting material required was
considerably in excess of what we could hope to purify from our
in vitro assay system (subnanogram from a single assay; see
"Experimental Procedures" for the basis of this estimate). We opted
instead for a biochemical approach relying on a comparison between VSG
hydrazide and another VSG reporter expressed in procyclic trypanosomes.
This reporter, 117
Fig. 6 (lanes 1 and
2) shows that VSG hydrazide and 117 In this paper we describe a convenient assay for GPI anchoring
based on a cell-free system from insect stage African trypanosomes engineered to express a well characterized GPI-anchored protein. We use
the assay to demonstrate explicitly that the enzyme-mediated, hydrazine-induced cleavage of the GPI signal sequence from a
pro-protein results in the formation of a soluble protein hydrazide
product. As illustrated in Fig. 7, the reaction most likely proceeds
via activation of the carbonyl group of the In setting up our assay, we chose to work with procyclic stage African
trypanosomes because the more commonly used bloodstream stage cells
possess a membrane-associated GPI-hydrolyzing phospholipase C
activity (30) that would have confounded our experimental readout
(3). Due to the nonavailability of adequate immunological reagents and
the lack of cysteine/methionine residues for convenient metabolic
radiolabeling in procyclin the major GPI-anchored protein of procyclic
trypanosomes (31, 32), we focused instead on procyclic lines that had
been engineered to express the more experimentally accessible
bloodstream form VSG (14).
Incubation of pulse-labeled procyclic membranes with hydrazine results
in the release of VSG-hydrazide, which is recovered in the supernatant
of a carbonate wash of the membranes. Despite the brevity of the
labeling pulse during which most of the labeled VSG would be expected
to be ER-localized (14), the bulk of the labeled VSG appears not to be
accessible to the transamidase, and the amount of released VSG is only
a small fraction of the radiolabeled VSG still bound to the membranes
(data not shown). This small accessible fraction is eliminated
altogether when the labeled cells are "chased" (Fig.
2A), consistent with processing of VSG molecules and export
from the ER during the chase period. The small size of the releasable
fraction of pulse-radiolabeled VSG can be explained by proposing that
VSG pro-protein, not GPI-anchored VSG, is a substrate for the
transamidase. Although GPI-anchored VSG could, in principle, be a
substrate for the transamidase, we have no data to show that this is
the case. The pro-protein pool is expected to be small under normal
conditions, and the amount of VSG hydrazide formed is a reflection of
the endogenous pro-VSG pool size. The extent to which pro-VSG can be
processed may be further restricted if the transamidase is confined to
an ER domain, possibly in proximity to ER protein translocons (33).
Our overall objective in initiating these studies was to provide
biochemical proof of the anchoring mechanism and also to set the stage
for subsequent attempts to purify the enzyme. The GPIT has not been
purified, and its precise polypeptide make-up is unknown. Genetic
approaches in yeast and mammalian cells have led to the identification
of two distinct gene products (Gaa1p and Gpi8p) that are required for
GPIT activity, possibly implying that GPIT is a complex of at least two
polypeptides (34-36)). Gpi8p is homologous to a jackbean endopeptidase
that is involved in a transpeptidation reaction required for the
post-translational processing of concanavalin A (35, 37), and evidence
from the mammalian system suggests that Gpi8p may be responsible for
the transamidase activity of GPIT (36). However, it is unclear whether Gpi8p is the transamidase itself (35) or whether it requires co-factors
such as Gaa1p for activity. The function of Gaa1p is unknown, although
it may act to anchor the putative GPIT complex in the ER (34). In
analogy with other ER enzymes involved in the co- and
post-translational modifications of translocated proteins (38, 39),
GPIT may well be a complex of several polypeptides, including Gaa1p and
Gpi8p. Assignment of GPIT activity to a particular ER polypeptide (or
protein complex) would ultimately require purification of the enzyme
and reconstitution of the enzymatic activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gpi) have been
described previously (14). Using standard polymerase chain reaction and
cloning procedures (15), a peptide transmembrane domain-anchored VSG
construct (117tm) was created in which the transmembrane domain of a
Trypanosoma brucei lysosomal membrane protein (16), was
fused to the
amino acid of VSG 117, thereby replacing the GPI
anchor addition signal. The C-terminal amino acid sequence of this
construct is: ...
CKDASRSTGIIAVVAALVVGVIAVVLMRPRRRstop, where the
amino acid is in bold and the hydrophobic domain is underlined. A
stable procyclic cell line expressing this construct was generated as
described for the 117wt reporter. [35S]Cys/Met metabolic
radiolabeling of procyclic cell lines was carried out as described
previously (14) except that cells were labeled at a density of
108/ml. Cell viability was monitored throughout the
labeling period and was consistently >99%.
70 °C.
GPI (a VSG molecule
lacking the C-terminal GPI signal sequence) was analyzed alongside as a
control. Procyclic trypanosomes expressing 117
GPI were metabolically
labeled with [35S]Cys/Met as described, and lysates were
prepared. The lysates were supplemented with TEN-D buffer (without
prior washing) and taken for immunoprecipitation with anti-VSG
antibodies preadsorbed to PAS beads as above. The immunoprecipitated
117
GPI was analyzed by carboxypeptidase treatment as described for
VSG hydrazide.
Ser (a gift from Dr. Sidney Udenfriend)) was
translated using a nuclease-treated rabbit reticulocyte lysate in the
presence of rough microsomes (prepared from mouse thymoma cells
(BW5147.3)) according to previously published procedures (12).
[35S]Methionine was included in the translation reaction
to label the translation product. Translation-translocation was allowed to proceed for 30 min at 27 °C before adding hydrazine,
p-chloromercuriphenylsulfonic acid (pCMPSA),
p-chloromercuribenzoate (pCMB), or iodoacetamide as
indicated and continuing the incubation for an additional 90 min. The
samples were then treated with proteinase K (85 µg/ml final
concentration) for 20 min on ice, diluted with buffer containing phenylmethylsulfonyl fluoride, and centrifuged to recover the membranes
as described (12). The membrane pellet was resuspended in
SDS-containing sample buffer and analyzed by SDS-PAGE and fluorography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Production of carbonate-extractable VSG on
incubation of metabolically pulse-labeled procyclic trypanosome lysates
with hydrazine or hydroxylamine. A, crude membranes
from [35S]Cys/Met pulse-radiolabeled VSG 117-expressing
procyclic trypanosomes were incubated in the absence or presence of
hydrazine or hydroxylamine as described under "Experimental
Procedures." Reactions were carried out at 37 °C for 0, 20, or 40 min as indicated. The samples were then analyzed by sodium carbonate
extraction, immunoprecipitation with anti-VSG 117 antibodies, and
SDS-PAGE and fluorography. B, crude membranes were incubated
with hydrazine for 45 min at different temperatures as indicated and
processed as described for A. Released VSG was quantitated
by densitometry of the SDS-PAGE fluorogram. Hz, hydrazine,
Hx, hydroxylamine.
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Fig. 2.
GPIT is located early in the secretory
pathway and cannot process a transmembrane form of VSG.
A, hydrazine-stimulated VSG release was assayed in crude
membranes prepared from trypanosomes pulse-labeled with
[35S]Cys/Met for 15 min (Pulse) or
pulse-labeled for 15 min and chased (in cysteine-methionine-containing
medium) for 1 h (Pulse-Chase). Samples were analyzed as
in Fig. 1A. B, crude membranes were prepared from
[35S]Cys/Met-labeled procyclic trypanosomes expressing
VSG anchored via a conventional transmembrane domain (see
"Experimental Procedures"). The membranes were incubated with or
without hydrazine as described under "Experimental Procedures," and
both the sodium carbonate extracted supernatant and pellet from the
incubations (as indicated) were immunoprecipitated and analyzed by
SDS-PAGE/fluorography. Hz, hydrazine.
1-2Man
1-6Man
1-4GlcN
1-6Inos(acyl)-PO4-monoacylglycerol), as well as the PP1 precursor, PP3
(ethanolamine-PO4-Man
1-2Man
1-6Man
1-4GlcN
1-6Inos(acyl)-PO4-diacylglycerol) (25, 26) as shown by thin layer chromatographic analysis (Fig. 3A, panel I). Both
lipids are susceptible to hydrolysis by GPI-specific phospholipase D
(Fig. 3A, compare panels I and II).
When cells were labeled in the presence of mannosamine, GPI synthesis
was almost completely abolished: no
[3H]ethanolamine-labeled PP1 was detected, and only a
trace amount of PP3 was synthesized (Fig. 3A, panels
III and IV).
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Fig. 3.
Pro-VSG is a substrate for GPIT.
A, crude membranes were prepared from VSG 117-expressing
procyclic trypanosomes metabolically labeled with
[3H]ethanolamine in the absence or presence of
mannosamine (ManN). Lipids from 1 × 108
cell equivalents of membranes were extracted into organic solvent
(chloroform:methanol:water, 10:10:3 v/v/v). The lipid extract was dried
under a stream of nitrogen, and the residue was partitioned into the
upper phase of an n-butanol:water two-phase mixture. Lipids
recovered in the upper n-butanol-rich phase were dried under
nitrogen, resuspended in detergent-containing buffer as described under
"Experimental Procedures," and treated with or without GPI-specific
phospholipase D as indicated. Lipids were then re-extracted and
analyzed by high performance TLC as described under "Experimental
Procedures." The chromatograms were visualized using a TLC
radioscanner. A segment of the chromatogram (corresponding to the
region containing the GPIs PP1 (*) and PP3 ( ) is shown.
B, 2.5 × 107 cell equivalents of
[3H]ethanolamine-labeled washed trypanosome membranes
(prepared from cells labeled in the absence or presence of mannosamine)
were resuspended in buffer for immunoprecipitation (TEN-D) and
incubated with anti-VSG 117 antibodies. The immunoprecipitates were
analyzed by SDS-PAGE/fluorography, and laser densitometry was used to
quantitate the amount of [3H]ethanolamine-labeled
proteins. C, crude membranes prepared from cells
pulse-labeled with [35S]Cys/Met for 15 min in the absence
or presence of mannosamine were incubated with or without hydrazine for
45 min as in Fig. 1A and analyzed by carbonate extraction,
immunoprecipitation, and SDS-PAGE/fluorography. Hz,
hydrazine.
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Fig. 4.
Inhibition of trypanosome (but not mammalian)
GPIT with sulfhydryl alkylating reagents. A,
hydrazine-stimulated release of VSG was assayed in the presence of
sulfhydryl alkylating reagents pCMPSA (lane 4), pCMB
(lane 6), and iodoacetamide (lane 5). Both the
pCMPSA and the pCMB incubations contained a final concentration of 4 mM NaOH (used to dissolve the reagents); control assays in
the presence of 4 mM NaOH are shown in lanes 3 and 7. B, GPI anchoring using mammalian cell
microsomes and the prepromini-PLAP reporter was assayed as described
under "Experimental Procedures." Membranes were preloaded with
promini-PLAP (lanes 1, 3, 5, and
7) and then incubated with hydrazine (lanes 2,
4, 6, and 8) in the absence
(lane 2) or presence (lanes 4, 6, and 8) of
sulfhydryl alkylating reagents. Samples were processed by proteinase K
treatment (to strip untranslocated prepromini-PLAP),
immunoprecipitation, SDS-PAGE, and fluorography. Incubations with 4 mM NaOH (used as a solvent for pCMPSA and pCMB) yielded
results identical to those shown in lanes 1 and
2.
site in mini-PLAP) during the
30-min loading period and that treatment with sulfhydryl alkylating
reagents during the "second" incubation is unlikely to have an effect.
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Fig. 5.
GPIT-mediated biotinylation of VSG in the
presence of biotin-LC-hydrazide. A, 5 × 107 cell equivalents of [35S]Cys/Met
pulse-labeled trypanosomes were used for the incubations in lanes
1-4. Washed membranes were incubated in the absence (lane
2) or presence of hydrazine (lane 1) or
biotin-LC-hydrazide (lane 4). The biotin-LC-hydrazide was
dissolved in dimethyl sulfoxide (DMSO); a control incubation
with just Me2SO added was also performed (lane
3). The sodium carbonate extract supernatant from these
incubations was processed by washing on a Millipore filter, dissolved
in buffer, and immunoprecipitated with anti-VSG 117 antibodies. The
immunoprecipitates (IP) were analyzed by SDS-PAGE and
fluorography (lanes 1-4). An identical set of incubations
was also performed on an 8-fold larger scale, and the
immunoprecipitates obtained using anti-VSG 117 antibodies were then
subjected to precipitation with streptavidin agarose as described under
"Experimental Procedures." The streptavidin-bound material was
analyzed by SDS-PAGE/fluorography (lanes 5-8).
B, purified [3H]myristic acid-labeled
GPI-anchored VSG 117 (500 cpm) was biotinylated using sulfo-NHS-biotin
or biotin-LC-hydrazide as described under "Experimental
Procedures." The treated material was subjected to
streptavidin-agarose precipitation, and the precipitates were taken for
scintillation counting. The [3H] cpm recovered are shown
as the means ± error of duplicate determinations. Hz,
hydrazine.
GPI, is a VSG 117 molecule lacking the C-terminal
GPI signal sequence. VSG hydrazide and 117
GPI should be similar
except for the hydrazine residue covalently incorporated into VSG
hydrazide (Fig. 5A). A transamidation reaction mechanism
(see Fig. 7) predicts that hydrazine will be incorporated at the C
terminus of the released VSG hydrazide molecule rendering it
insusceptible to attack by carboxypeptidases. In contrast, 117
GPI
with an unblocked C terminus should be susceptible to proteolysis by carboxypeptidases.
GPI appear to be of
identical molecular mass, consistent with the proposal that VSG
hydrazide represents a full-length VSG molecule truncated at or close
to the
site. The results of carboxypeptidase treatment are shown in
Fig. 6 (lanes 3 and 4). The data show clearly that although 117
GPI can be proteolyzed by carboxypeptidases (compare lanes 2 and 4), VSG hydrazide resists
proteolysis (compare lanes 1 and 3). These data
are consistent with the proposal that the C terminus of VSG hydrazide
is blocked by hydrazine as expected for the product of the
transamidation reaction illustrated in Fig.
7.
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Fig. 6.
Hydrazine is incorporated at or near the C
terminus of GPIT-released VSG hydrazide. VSG 117 hydrazine
(Hz) (obtained as in Fig. 1A) and 117 GPI
(isolated from procyclic trypanosomes expressing this construct) were
purified by immunoprecipitation and analyzed by SDS/PAGE fluorography
before (lanes 1 and 2) or after (lanes
3 and 4) carboxypeptidase treatment. Both molecules run
identically before carboxypeptidase treatment (lanes 1 and
2), but only 117
GPI is proteolyzed after incubation with
a mixture of carboxypeptidases P and W.
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Fig. 7.
Proposed mechanism for the
transamidase-mediated, nucleophile-induced release of VSG (modified
from Ref. 1). The carbonyl group of the amino acid (aspartic
acid) of pro-VSG is activated by a sulfhydryl group in the transamidase
(Enz-S
) resulting in the formation of an
enzyme-substrate complex and cleavage of the amide bond between
aspartic acid and serine (
+ 1 in the c-terminal signal sequence of
pro-VSG). Nucleophilic attack by H2N-X results in release
of VSG-NH-X and regeneration of the active site sulfhydryl in the
transamidase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
residue of the
pro-protein (or GPI protein) by a hydrophilic group on the
transamidase, with concomitant displacement of the GPI signal sequence
(or GPI anchor). We provide direct evidence that the pro-protein is
indeed a substrate in our assay by using membranes prepared from cells
blocked in GPI biosynthesis (Fig. 3). The ability of sulfhydryl
alkylating reagents to abolish the reaction is consistent with the
proposal that the catalytic residue in the enzyme is an -SH group; the enzyme is accordingly depicted as Enz-S
in Fig. 7.
Nucleophilic attack on the activated carbonyl group by hydrazine
regenerates the enzyme and yields a protein hydrazide product. We show
that the protein product of the reaction is a protein hydrazide by
using the hydrazine derivative, biotin-LC-hydrazide, and specific
precipitation with streptavidin beads (Fig. 5A, lane 8). Characteristics of the assay (Figs. 1 and 2) as well as the molecular mass of the released material and its resistance to proteolysis by carboxypeptidases (Fig. 6) indicate that hydrazine is
incorporated into VSG at the
site (see "Results"). This result strengthens the proof, initiated by the work of Mayor et al.
(3) and Maxwell et al. (4), that GPI anchoring proceeds via
a transamidation reaction mechanism. Precedent for the type of analysis
described above may be found in studies of
-glutamyl transpeptidase.
Here a
-glutamyl-enzyme intermediate is formed that undergoes
nucleophilic attack by an amino acid acceptor (to form a
-glutamyl-amino acid) or water (to form glutamate) (28).
Importantly, for the purposes of the work presented in this paper,
-glutamyl transpeptidase can catalyze reactions between
-glutamyl
compounds and nucleophiles (e.g. hydroxylamine) to generate
the corresponding
-glutamyl derivatives (e.g.
hydroxamates) (29).
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ACKNOWLEDGEMENTS |
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We thank Dawn Ransom and Vivian Fu for help with trypanosome cultures, Venera Bouriakova for carrying out preliminary experiments, Angela Mehlert for help with the mass spectrometric analyses, Andreas Conzelmann, Terry Smith, Mike Ferguson, Jitu Mayor, and Peter Bütikofer for helpful discussions, and Dave Rancour, Niki Baumann, and Cedric Simonot for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM55427 (to A. K. M.) and AI35739 (to J. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Wellcome Prize Travelling Research Fellowship from the Wellcome Trust. To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706-1544. Tel.: 608-263-2636; Fax: 608-262-3453; E-mail: dsharma{at}biochem.wisc.edu.
International Scholar of the Howard Hughes Medical Institute.
Burroughs Wellcome Fund New Investigator in Molecular Parasitology.
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ABBREVIATIONS |
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The abbreviations used are: GPI, glycosylphosphatidylinositol; GPIT, GPI transamidase; PAS, protein A-Sepharose; pCMB, p-chloromercuribenzoate; pCMPSA, p-chloromercuriphenylsulfonic acid; PLAP, placental alkaline phosphatase; PP1, procyclic GPI anchor precursor; PP3, procyclic GPI (precursor to PP1); VSG, variant surface glycoprotein; PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum.
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