From the Departments of Biochemistry, Pediatrics and Cell Biology and Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
Received for publication, October 24, 2002, and in revised form, November 10, 2002
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ABSTRACT |
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In liver, phosphatidylethanolamine is
converted to phosphatidylcholine through a series of three sequential
methylation reactions. Phosphatidylethanolamine
N-methyltransferase (PEMT) catalyzes each
transmethylation reaction, and
S-adenosylmethionine is the methyl group donor.
Biochemical analysis of human liver revealed that the methyltransferase
activity is primarily localized to the endoplasmic reticulum and
mitochondria-associated membranes. Bioinformatic analysis of the
predicted amino acid sequence suggested that the enzyme adopts a
polytopic conformation in those membranes. To elucidate the precise
membrane topography of PEMT and thereby provide the basis for in-depth
functional characterization of the enzyme, we performed
endoproteinase-protection analysis of epitope-tagged, recombinant
protein. Our data suggest a topographical model of PEMT in which four
transmembrane regions span the membrane such that both the N and C
termini of the enzyme are localized external to the ER. Two hydrophilic
connecting loops protrude into the luminal space of the microsomes
whereas a corresponding loop on the cytosolic side remains proximate to
the membrane. Further support for this model was obtained following
endoproteinase-protection analysis of mutant recombinant PEMT
derivatives in which specific protease cleavage sites had been
genetically engineered or ablated.
All eukaryotic cells synthesize phosphatidylcholine
(PC)1, which has an integral
role in membrane ultrastructure and intracellular signaling (1, 2). In
hepatocytes, an additional and substantial demand is imposed on the PC
pool by the liver-specific functions of bile and very low density
lipoprotein (VLDL) particle production and secretion (3, 4). The
phosphatidylethanolamine N-methyltransferase (PEMT) and
CDP-choline biosynthetic pathways mediate continual replenishment of
the hepatic PC pools (5). The liver is the primary site of PEMT
activity whereas the enzymes of the CDP-choline pathway are active in
all nucleated cells (2, 6).
PC biosynthesis is clearly essential to liver function, but why that
synthesis must be conducted through two distinct pathways is less
evident. Recent studies investigating the proportion of hepatic PC that
is derived from each pathway defined the PEMT-controlled pathway as the
source of 30% of hepatic PC with the CDP-choline pathway accounting
for 70% (7-9). Significantly perhaps, data from one group also
revealed that the PEMT pathway is a metabolically channeled process
suggesting that PEMT-derived PC may be destined for a specific function
(9).
Given that PEMT is primarily expressed in liver, PEMT-derived PC might
be targeted to a liver-specific fate such as VLDL particles or bile
(10-13). In efforts to address these hypotheses, studies were recently
conducted using hepatocytes from mice homozygous for a disrupted PEMT
allele. The data revealed a defect in the secretion of triacylglycerol
and apo B100, key components of VLDL particles, suggesting that PEMT is
required for optimal VLDL assembly and/or secretion (14). Additional
studies investigating a role for PEMT in bile production or secretion
are currently in progress.
Metabolic channeling is central to several metabolic processes
including glycolysis and glycogenolysis and involves the retention of
metabolites in a specific microenvironment to promote consecutive enzymatic reactions and hence efficient energy utilization (15). For
metabolic channeling to be effective, however, spatial organization is
requisite. Thus, not only should enzymes and substrates be localized in
the same cellular subcompartment, but enzymes must also be
topographically organized such that key catalytic residues or motifs
are correctly oriented.
Liver is the primary site of human PEMT expression, with extra-hepatic
PEMT accounting for a mere fraction of corporeal expression (6,
16-20). Herein, biochemical analysis of human liver reveals that PEMT
is primarily localized to the endoplasmic reticulum (ER) and a
subfraction of ER membranes that co-fractionate with mitochondria,
mitochondria associated membranes (MAM). However, the exact topography
of the enzyme within those membranes has not been determined.
Resolution of the topographical orientation of PEMT will permit further
analysis of the role of metabolic partitioning in PC biosynthesis.
Moreover, it will provide the basis for in depth exploration of the
mechanism by which PEMT becomes rate-limiting in the secretion of VLDL particles.
Bioinformatic analysis predicts that PEMT is a polytopic membrane
protein with four transmembrane domains and thus yields a model that
positions the N and C termini of the enzyme in the same intracellular
compartment (21). However, in silico analysis cannot resolve
whether the end termini of PEMT reside in the cytosol or in the
microsomal lumen.
Here, we detail the biochemical validation of a topographical model of
PEMT, using endoproteinase protection analysis of functional epitope-tagged derivatives of the enzyme. These studies will provide the basis for detailed structural and functional characterization of
the human enzyme.
Materials--
Dulbecco's modified Eagle's medium, fetal
bovine serum, restriction endonucleases, and Platinum Pfx DNA
polymerase were from Invitrogen. Oligonucleotides for
mutagenesis and epitope tagging were synthesized at the DNA core
facility in the Department of Biochemistry, University of Alberta.
FuGENE transfection reagent was from Roche Molecular Biochemicals.
S-Adenosyl-L-[methyl-3H]methionine
(15 Ci/mmol) was obtained from Amersham Biosciences. Non-radiolabeled
S-adenosyl-L-methionine, anti-HA monoclonal
antibody (clone HA-7), and endoproteinase Lys-C were from Sigma. Rabbit polyclonal anti-protein disulfide isomerase (PDI) antibody was from
Stressgen Biotech. Goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Pierce. All other reagents were of the
highest standard commercially available.
Subcellular Fractionation--
Adult human liver samples were
obtained from the Department of Surgery at the University Of Alberta
Hospital and were snap-frozen in liquid nitrogen at resection.
Differential subcellular fractionation was performed according to the
procedure of Croze and Morre (22) as modified by Vance (23), yielding
fractions corresponding to ER, nucleus, plasma membrane, mitochondria,
MAM, and Golgi apparatus. Protein concentrations of individual
fractions were measured by the Bradford method, using albumin as standard.
Microsomes for protease protection analysis were prepared by a modified
version of the method of Graham (24). Briefly, 24 h
post-transfection with the various human PEMT (hPEMT) recombinant plasmids, COS-7 cells were washed, harvested into phosphate-buffered saline, and pelleted at 1000 × g. The pellet was
resuspended in Buffer A (50 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA), sonicated for 5 s,
and centrifuged at 6000 × g for 15 min to pellet
nuclei, heavy mitochondria, plasma membrane, Golgi, and cell debris.
The resulting supernatant was then centrifuged for 45 min, at
99,000 × rpm at 4 °C. The pellet was resuspended in 75 µl of
Tris-buffered saline by pipetting gently 20 times. Protein
concentrations of the prepared microsomes were determined by the
Bradford method. Integrity of the microsomes was verified by
immunoblotting with a polyclonal antibody against the ER luminal
marker, PDI.
Bioinformatic Analysis--
Hydropathy analysis, based on the
method of Kyte and Doolittle (25) was performed on the predicted human
PEMT amino acid sequence (GenBankTM accession number
NP_009100), using the Grease program of the San Diego Supercomputer
Center Biology Workbench (workbench.sdsc.edu). The TMAP program in the
Biology Workbench was employed to predict the position and length of
individual transmembrane domains (26).
Recombinant Plasmid Construction--
All plasmids were
constructed using the wild-type hPEMT-pCI plasmid as template (21).
This plasmid consists of the human PEMT open reading frame cloned 5' to
3' into the XhoI and XbaI sites, respectively, of
the pCI mammalian expression vector polylinker (Promega). Transcription
is under the control of a cytomegalovirus promoter. Using PCR, an
oligonucleotide encoding an HA (YPYDVPDYA)-tagged epitope was appended
to the 5' end of hPEMT to generate the plasmid, HA-hPEMT. PCR products
were blunt-end ligated into SmaI-cut pBluescript II (KS)
(Stratagene) and recloned to pCI using XhoI and
XbaI restriction sites. Mutant PEMT derivatives for protease
protection analysis were generated by the "splice by overlap
extension" PCR mutagenesis method, using the HA-hPEMT plasmid
as template (27). Full-length mutant products were subcloned into the
pCI expression vector as detailed above. All constructs were sequenced
to confirm fidelity of PCR and orientation of the insert at the
Molecular Biology Services Unit, Department of Biological Sciences,
University of Alberta. To mutagenize the two lysines at positions 38 and 41 in loop A to arginine residues, generating the HA-tagged double mutant HA-AK2R2, PCR A was performed with oligonucleotides 1 (5'-CTCGAGATGTATCCATATGATGTTCCAGATTATGCTACCCGGCTGCTGGGCTACGTGGACCCCCTG-3') and 2 (5'-TGTTCCCATCGTGCAACCACATTCCAG-3'), PCR B was performed with
oligonucleotides 3 (5'-GTGGTTGCACGATGGGAACACAGGACCCGCAGGCTGAGCAGGGCCTTCG-3') and 4 (5'-TCTAGATCAGCTCCTCTTGTGGGACCCGGAGGCT-3'), and PCR C, to generate the
full-length mutant product, was performed with oligonucleotides 1 and
4, using amplicons from PCR A and B as templates. To mutate the two
C- terminal lysines at positions 191 and 197 to arginine residues and
generate the HA-tagged double mutant HA-CK2R2, PCR D was performed with
oligonucleotides 1 and 5 (5'-TCTAGATCAGCTCCTCCTGTGGGACCCGGAGGCTCTCTGCCG-3'). To insert a novel
endoproteinase Lys-C cleavage site into loop B, the arginine
residue at position 80 was mutated to a lysine, generating the plasmid
HA-R80K. This was achieved as follows: PCR E was performed with
oligonucleotides 1 and 6 (5'-GGCTGGCTCAGCATGGCCTG-3'), PCR F was
performed with oligonucleotides 7 (5'-CAGGCCATGCTGAGCCAGCCCAAGATGGAGAGCCTGGAC-3') and 4, and the
full-length product, using PCR products E and F as template, was
generated using oligonucleotides 1 and 4. Each full-length mutant
amplicon was recloned into pCI as described.
Cell Culture and Transfections--
COS-7 cells, obtained from
the American Type Culture Collection repository, were maintained in
Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate at 37 °C,
5% CO2. On day 0, cells were plated at a confluency of
1.75 × 106 cells/60-mm dish. After an overnight
incubation, cells were transfected with test plasmids or mock
transfected with empty pCI expression vector using FuGENE transfection
reagent as per the manufacturer's protocol. Specifically, we used 5 µl FuGENE/3 µg DNA plasmid per 60-mm dish. Cells were harvested
24 h later and treated as described in the figure legends.
Phosphatidylethanolamine N-Methyltransferase Activity
Assays--
PEMT activity assays were performed as described
previously (28). Briefly, 24 h after transfection, COS-7 cells
were washed with and harvested into phosphate-buffered saline, pelleted
at 1000 × g, and resuspended in Buffer B (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM
EDTA). Following homogenization by sonication, protein homogenates (50 µg) were assayed for PEMT activity using
phosphatidylmonomethylethanolamine (Avanti Polar Lipids, Alabaster, AL)
as a methyl acceptor and S-adenosyl-L-[methyl-3H]methionine
as the methyl group donor.
Immunoblot Analysis--
Cell homogenate proteins (25 µg) were
separated by Tris/glycine SDS-polyacrylamide electrophoresis, on 12.5%
polyacrylamide gels calibrated with prestained molecular weight
standards (Bio-Rad). Following electrophoresis, proteins were
transferred to PVDF membranes and immunoblotted with primary antibodies
at the indicated concentration. Protein-antibody complexes were
detected by enhanced chemiluminescence with horseradish
peroxidase-conjugated secondary antibody using the ECL reagent
(Amersham Biosciences) as directed. Membranes were exposed to Biomax MR
film (Eastman Kodak Co.) for the indicated time at room temperature.
Endoproteinase Lys-C Protection Assays--
Endoproteinase
protection analyses were performed on microsomes prepared from
transfected COS-7 cells as described above. Briefly, microsomal
proteins (50 µg) were incubated with 1% Triton X-100 or 100 mM Tris-HCl, pH 8.5, on ice for 30 min. Endoproteinase Lys-C (0, 0.1, or 1 µg) was added to the mixture (final reaction volume, 20 µl) and incubated at 37 °C for 3 h. Each reaction
was stopped by the addition of Buffer C (5× 60 mM
Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 715 mM
Subcellular Localization of PEMT--
Early studies on human PEMT
identified the liver as the primary site of expression, which parallels
the expression pattern of PEMT in rodents (6, 29). Although the rat
PEMT activity is distributed between ER and MAM, only the isoform
designated PEMT2, in the MAM fraction, is immunoreactive with an
antibody raised against a C-terminal rat PEMT peptide (16). To
determine whether the human enzyme displays similar disparity in the
localization of enzymatic activity and immunoreactivity, subcellular
fractionation of human liver was performed. Similar to rodents, the
human PEMT activity is primarily localized to ER and MAM (Fig.
1A), but unlike rodents the
human PEMT enzyme is also immunoreactive to the anti-PEMT peptide
antibody in both ER and MAM (Fig. 1B). Immunoblotting with
anti-PDI confirmed that the fractions representing ER and MAM (which is
a subfraction of ER) were of ER origin (Fig. 1C). As the
activity and immunoreactivity of human PEMT are superimposable, it
appears that the differential subcellular localization of PEMT isoforms, as observed in the rat, has not been conserved in
evolution.
Predicted Membrane Topography of PEMT--
Purification of PEMT
revealed the enzyme to be an integral membrane protein (30). To gain
insight into the topography of PEMT in the membranes of ER/MAM, the
deduced amino acid sequence was examined in silico using the
method of Kyte and Doolittle (25). The hydropathy profile of PEMT shown
in Fig. 2A predicts the
presence of four hydrophobic regions. Each hydrophobic region exceeds
20 amino acids in length and registers a value of >2 units on the
hydropathy plot, two properties strongly indicative of a transmembrane
domain. A polytopic model based on four transmembrane
To investigate which of the two possible topographical models is valid,
intact microsomes were prepared from transfected cells and subjected to
endoproteinase digestion in the absence or presence of detergent. In
the absence of detergent, proteolysis is expected to occur only at
exposed cleavage sites on the microsomal exterior, and the luminally
oriented sites should remain protected. Cleavage of protected luminally
oriented sites should occur only in the presence of detergent. The
protease utilized in these studies, endoproteinase Lys-C, specifically
cleaves at the C terminus of lysine residues, and, given the position
of the lysine residues within the PEMT amino acid sequence, was
expected to yield an informative proteolytic pattern (Fig. 2,
B and C).
Characterization of HA-tagged PEMT Protein--
To perform the
topographical analyses, an antibody capable of detecting proteolytic
cleavage products was required. An antibody against a peptide
corresponding to an epitope at the extreme C terminus of PEMT was
raised previously, but this epitope contains two lysine residues
(endoproteinase Lys-C cleavage sites) with the consequence that
proteolysis would result in cleavage at these residues and destruction
of the epitope. Therefore, although this antibody is informative for
the localization of the C terminus, it would not be expected to yield
interpretable results for the remainder of the protein.
To circumvent this problem a HA tag, which does not contain lysine
residues, was appended to the N terminus of PEMT. Hydropathy analysis
of the epitope-tagged protein sequence did not predict changes in the
number or length of the predicted transmembrane domains. Furthermore,
the HA-tagged PEMT expressed in COS-7 cells is enzymatically active
(Fig. 3A). Immunoblotting
verified the production of recombinant proteins (Fig. 3B)
and faithful recognition of the HA antigen tag by the anti-HA antibody
(Fig. 3C).
Protease Protection Analysis of Epitope-tagged PEMT--
To
analyze which one of the two possible membrane topographic models of
PEMT is valid, a plasmid encoding HA-tagged PEMT was transfected into
COS-7 cells. Subsequently, microsomes were prepared and incubated with
various concentrations of endoproteinase Lys-C in the absence or
presence of Triton X-100, and the resultant proteolytic products were
separated by SDS-polyacrylamide gel electrophoreses and analyzed by
immunoblotting (Fig. 4A). In
microsomes incubated without protease, an immunoreactive band
corresponding to the epitope-tagged PEMT was detectable at ~22 kDa in
the absence or presence of Triton X-100 (Fig. 4A,
lanes 1 and 2).
In the presence of endoproteinase, but in the absence of Triton X-100,
the ~22-kDa band was replaced by one of increased electrophoretic mobility (Fig. 4A, lanes 3 and 5).
This is indicative of cleavage at the C-terminal lysine residues, which
generates a truncation product lacking the final eight residues of the
epitope-tagged protein. As proteolysis occurred in the absence of
detergent, this result suggests that the C terminus of PEMT is
localized external to the microsomes.
To confirm that the endoproteinase was functional in the absence of
Triton X-100, and hence that the electrophoretic shift observed in Fig.
4A (lanes 3-6) was because of proteolytic
cleavage, duplicate proteolytic products were immunoblotted with a
rabbit polyclonal anti-PEMT antibody. Protease treatment, in the
absence or presence of Triton X-100, resulted in destruction of the
C-terminal PEMT epitope and consequently, a loss of immunoreactivity,
confirming that the protease remained active (Fig. 4B,
lanes 3-6).
In the presence of Triton X-100, the C-terminal truncation product was
again evident, but a reduction in intensity resulted as protease
concentrations increased (Fig. 4A, lanes 4 and
6). This was because of proteolysis at the previously
inaccessible luminal cleavage sites. Digestion in the presence of
Triton X-100 also resulted in the appearance of a fast migrating band
of ~5.2 kDa (lanes 4 and 6). This band
corresponds to the expected proteolytic product resulting from cleavage
at the lysine residues in loop A. The diffuse appearance of the 5.2-kDa
immunoreactive band is probably because of the high concentration of
proteolytic fragments in this region of the gel. Given the appearance
of this immunoreactive band only in the presence of detergent, loop A
appears to reside in the ER lumen.
To verify the integrity of the prepared microsomes, duplicate
proteolysis products were immunoblotted with an antibody against the ER
luminal marker, PDI (Fig. 4C). In the absence of detergent, a 57-kDa immunoreactive band was detectable, indicating protection of
the epitope and thus demonstrating the integrity of the microsomes (Fig. 4C, lanes 1, 3, and
5). In the presence of detergent, proteolysis abolished the
immunoreactivity of PDI, demonstrating that the detergent permeabilized
the microsomes and that the protease was active (Fig. 4C,
lanes 4 and 6). These data support the validity of a topographical model that localizes both termini external to the
microsomes (Fig. 4D).
Evaluation of HA-tagged PEMT Mutants--
Further analysis of the
proposed topography of PEMT required the design of three novel
HA-tagged PEMT derivatives. To confirm the specificity of cleavage at
the lysine residues in the C terminus (Fig. 4A), both
residues were mutated to arginine residues to generate the plasmid
HA-CK2R2. To evaluate the proposed cytosolic localization of loop B, a
mutant version of PEMT was generated in which a lysine residue and
hence endoproteinase site was engineered into loop B, resulting in the
plasmid HA-R80K. To confirm the specificity of cleavage at the two
lysine residues in loop A, and to investigate the orientation of loop
C, a third plasmid, HA-AK2R2, was generated in which both lysine
residues in loop A were mutated to arginine residues. To ensure that
the mutant constructs retained PEMT activity, each construct was
transfected into COS-7 cells, and activity assays were performed. Cells
transfected with each mutant construct displayed equal (HA-AK2R2) or
greater (HA-CK2R2, HA-R80K) PEMT activity compared with cells
expressing the unmodified PEMT enzyme (Fig.
5A), signifying that the
structure and topography required for enzymatic activity are retained.
Immunoblots demonstrated similar levels of expression of the
recombinant proteins (Fig. 5B).
Protease Protection Analysis of HA-tagged PEMT
Mutants--
In the next set of experiments, the plasmid HA-CK2R2,
encoding hPEMT, which lacks the C-terminal proteolysis sites (Fig.
4A), was transfected into COS-7 cells to evaluate the
specificity of endoproteinase cleavage at the C-terminal lysine
residues. Microsomes were prepared from the transfected cells, and
protease protection experiments were conducted as before. Digestion of
the microsomes with various concentrations of the protease, in the
absence of Triton X-100, did not change the electrophoretic mobility of
the ~22-kDa band that corresponds to the full-length tagged mutant protein (Fig. 6B, lanes
1-6). This contrasts with data obtained from the HA-hPEMT
proteolysis experiments (Fig. 4A, lanes 3-6), in
which the C-terminal lysine residues are intact, and cleavage results.
This result supports the notion that the C terminus of PEMT resides in
the cytosol, validating our earlier findings and one portion of our
predicted model (Fig. 4D). As anticipated, proteolytic
products from microsomes treated with protease in the presence of
detergent (lanes 4 and 6) were similar to those generated
from protection experiments on HA-hPEMT (Fig. 4A,
lanes 4 and 6). Reprobing of the membranes with a
polyclonal anti-PDI antibody verified the integrity of the microsomal
membranes (Fig. 6C).
The topographical model in Fig. 4D postulated that loop B is
exposed to the cytosol. To examine this hypothesis, COS-7 cells were
transfected with the plasmid HA-R80K, which contains an engineered endoproteinase site in loop B. Proteolysis in the absence of detergent was predicted to yield a novel immunoreactive proteolytic fragment reflecting cleavage at the exposed loop B site. However, cleavage did
not occur in the absence of detergent (results not shown), suggesting
that the engineered cleavage site is protected and that loop B is
localized proximate to the membrane. Given the length and
hydrophobicity of each predicted transmembrane domain, a bitopic model
based on two transmembrane domains that would orient loop B into the ER
lumen is unlikely. Thus, although the topography of loop B remains
indeterminate, a model positioning the hydrophilic connecting loop
contiguous with the external leaflet of the membrane bilayer is favored.
In the final set of experiments, the plasmid HA-AK2R2, in which the
cleavage sites in loop A are abolished (Fig.
7A), was transfected into
COS-7 cells, and protease protection analysis was performed. As
anticipated, results from the protease protection experiments conducted
in the absence of Triton X-100 (Fig. 7B, lanes 1,
3, and 5) were similar to those from similar
experiments on HA-hPEMT (Fig. 4A, lanes 1,
3, and 5). However, in the presence of detergent,
addition of protease failed to yield a proteolytic fragment of ~5.2
kDa (Fig. 7B, lanes 4 and 6),
confirming that the 5.2-kDa fragment generated following cleavage of
HA-hPEMT was a result of specific proteolysis at the lysine residues in loop A (Fig. 5B, lanes 4 and 6).
Hence, the predicted luminal localization of loop A is supported.
Furthermore, proteolysis in the presence of Triton X-100 yielded a
fragment of ~15 kDa as postulated. Previously, this product was not
generated because of the presence of the loop A cleavage sites within
the 15-kDa fragment. However, following ablation of the loop A sites,
the 15-kDa proteolytic product was generated following cleavage at the
lysine residue in loop C. Given that the appearance of this product
occurs only in the presence of detergent, our notion of a luminal
orientation for loop C is supported. Immunoblotting with a polyclonal
anti-PDI antibody confirmed the integrity of the microsomes and thus
the interpretation of our results.
Expression of the human PEMT gene is greatest in the
liver, and here we demonstrate that the encoded PEMT protein is
enriched subcellularly in both the ER and MAM (Fig. 1). This contrasts with findings in rats where two isoforms of PEMT exist that are distinguishable on the basis of immunoreactivity with an antibody raised against a rat PEMT C-terminal peptide; PEMT1 is localized to the
ER whereas PEMT2 is confined to the MAM (16). However, in humans, PEMT
activity and immunoreactivity are detectable in both the ER and MAM
suggesting that the differential subcellular localization of PEMT
isoforms may be confined to rodents (Fig. 1).
Although the localization of PEMT within the human hepatic
ultrastructure has now been revealed, factors that direct PEMT to the
specific subcellular compartment remain to be identified. Targeting of
ER membrane proteins is a well defined process that is modulated by
specific retention or retrieval signals (31, 32). Whereas the first
transmembrane segment of some polytopic proteins serves to
"retain" the protein in the ER membrane, a C-terminal
dilysine motif (KKXX or KXKXX) can
similarly confer ER localization, albeit through retrieval from an
intermediate compartment (31, 32). A C-terminal dilysine motif is
present in the yeast PEM2 amino acid sequence, but this motif is not
conserved in the higher eukaryotes. However, a hybrid
XHKRX motif is conserved in the rat, mouse, and
human amino acid sequences. Moreover, in certain instances, it has been
demonstrated that mutagenesis of one lysine in the dilysine motif to an
arginine or a histidine residue can occur without detriment to ER
targeting (33). Because histidine and arginine residues flank the
C-terminal lysine residue, one of several amino acid combinations could
potentially mediate ER targeting. For a dilysine motif to be functional
as an ER targeting signal, a cytosolic orientation is requisite. Our
proposed topographical model for PEMT in which the C terminus is
localized to the cytosol conforms to this requirement. Further analysis
would be required to determine the relative contribution of the
XHKRX motif to the subcellular distribution of
PEMT.
Elucidation of the subcellular distribution of the integral membrane
protein, PEMT, prompted an investigation of the topographical orientation of PEMT in the microsomal membranes. Although early trypsin-proteolysis studies suggested that certain domains of PEMT were
localized external to the microsomal membranes, the specific membrane
topography of PEMT had, until now, remained elusive (34). Here, we
present data that are consistent with the tetra-span membrane
topography model of PEMT shown in Fig. 2B.
Bioinformatic analysis of the PEMT amino acid sequence revealed the
presence of four regions of hydrophobicity that varied in length
between 23 and 29 amino acids. Separating the putative transmembrane
Because each hydrophobic region of the PEMT protein exceeds the minimum
length considered necessary for the formation of a transmembrane
segment (20 amino acids), and because of the relative hydrophobicity
(>2 units) of each segment, a membrane topography based on four
transmembrane domains is proposed (Fig. 2B) (37). In
contrast, whereas the yeast ortholog (PEM2) is proposed to be similarly
polytopic, one portion of the yeast protein contains a hydrophobic
stretch of 31 amino acids, which, intriguingly, is the minimum length
required for the formation of a helical hairpin (helix-turn-helix) in
the membrane (38). Moreover, a pair of residues with helix
turn-inducing propensity (lysine-proline) is centrally located in the
31-residue hydrophobic segment (39, 40). However, as each putative
transmembrane domain of the human protein ranges in length from 23 to
29 amino acids and is thus below the minimum requirement for the
formation of a helical hairpin, the four human transmembrane
Protease protection analysis of epitope-tagged PEMT in intact
microsomes revealed that the C terminus is sensitive to proteolytic digestion and hence is exposed to the cytosol, whereas both hydrophilic loops A and C are protease-resistant and are thus predicted to reside
in the lumen (see Fig. 4A, Fig. 6B, and Fig.
7B). Although the orientation of loop B and the N terminus
of PEMT were not resolved unequivocally in the present studies, a
hydropathy profile that is strongly indicative of a tetra-spanning
topography, combined with the orientation of loops A and C and the C
terminus, suggests that both loop B and the N-terminal domain are
cytosolically oriented. Furthermore, as the hydropathy profile of PEMT
is highly conserved in species from Rattus norvegicus to
Homo sapiens, the elucidated membrane topography of the
human enzyme should prove representative of the higher eukaryotic PEMT family.
Recent data from experiments utilizing isotopic labeling and NMR
spectroscopy suggest that channeling of metabolites occurs in the PEMT
pathway (9). Identification of residues essential for binding of the
methyl group donor, AdoMet, combined with the current data on the
subcellular localization and topographical orientation of PEMT, should
provide the clearest insight yet into the specific role of metabolic
channeling in this pathway.
Approximately 85% of methylation reactions occur in the liver, and
AdoMet is the primary methyl group donor (41, 42). Although several
consensus AdoMet binding motifs have been identified that are conserved
in the majority of AdoMet-dependent methyltransferases, a
small fraction of AdoMet-dependent methyltransferases,
including the eukaryotic PEMT family of enzymes, lack these motifs
(43). Cellular AdoMet is concentrated predominantly in the cytosol, with a smaller fraction present in mitochondria (44). Thus, we posit
that residues essential for binding of the AdoMet moiety are localized
in the cytosolically disposed hydrophilic loop (B) or at the cytosolic
face of the transmembrane In summary, we describe the first experimental resolution of the
topography of an enzyme that catalyzes the synthesis of PC. Data from
the current studies should provide the impetus for detailed structural
analysis of PEMT, which, in turn, should yield evidence for a
definitive topographical model of this AdoMet-dependent methyltransferase. Elucidation of the topographical organization of
PEMT will enable detailed analysis of the spatio-temporal organization of residues essential for the binding of AdoMet and hence promote a
mechanistic understanding of the methylation-dependent
biosynthesis of PC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 0.1% bromphenol blue) and boiled for 10 min.
Endoproteinase cleavage products were separated by Tris/glycine
SDS-polyacrylamide gel electrophoresis on 15% polyacrylamide gels and
immunoblotted as described above. Integrity of the microsomes was
validated by immunoblotting with a monoclonal anti-PDI antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Subcellular localization of PEMT in human
liver. Human liver samples, snap-frozen at resection, were
subjected to differential subcellular fractionation (22, 23).
A, PEMT specific activity in individual fractions.
Homogenates of each fraction, 50-µg protein, were assayed for PEMT
activity. PM, plasma membrane; Mito,
mitochondria. B, immunoblot with anti-PEMT antibody using 25 µg of protein homogenates for each fraction. C, immunoblot
with anti-PDI antibody using 25 µg of protein homogenates for each
fraction.
-helical
domains colocalizes the N and C termini on one side of the membrane
plane, suggesting that PEMT adopts one of two opposing topographical
orientations (both termini in the lumen or both termini in the
cytosol).
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Fig. 2.
Hydropathy plot and predicted membrane
topography of PEMT. A, hydropathy plot of the
human PEMT amino acid sequence, as determined by the Grease program
(based on the method of Kyte and Doolittle) (25), at the San Diego
Supercomputer Center Biology Workbench. B, working model for
PEMT topography. Hydrophilic connecting loops are labeled A,
B, and C. Arrows indicate
endoproteinase Lys-C cleavage sites. C, shaded and unshaded
regions in the PEMT amino acid sequence denote predicted transmembrane
-helices and hydrophilic connecting loops, respectively.
Asterisks indicate the position of lysine residues
(endoproteinase Lys-C cleavage sites).
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Fig. 3.
Epitope-tagged PEMT protein is enzymatically
active in transfected COS-7 cells. COS-7 cells were transiently
transfected with 3 µg of plasmids containing wild-type PEMT or
epitope-tagged PEMT derivatives, or they were mock transfected with
empty pCI vector. A, cellular homogenates, 50-µg protein,
were assayed for PEMT activity. The results are expressed as the mean
of three separate experiments, each performed in duplicate, ± S.E.,
relative to the values obtained for similar assays on cells transfected
with wild-type PEMT. B, immunoblot with anti-PEMT antibody
using 25-µg protein of transfected cellular homogenates.
C, immunoblot with anti-HA antibody using 25-µg protein of
transfected cellular homogenates.
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Fig. 4.
Luminal orientation of loop A and cytosolic
orientation of the C terminus as determined by protease protection
analysis. A, microsomes were prepared from transfected
cells as described under "Experimental Procedures." Aliquots,
50-µg protein, were incubated with various concentrations of
endoproteinase at 37 °C for 3 h, in the absence or presence of
1% Triton X-100. Reactions were stopped by the addition of
electrophoretic loading buffer and boiling at 100 °C for 10 min.
Samples were separated by SDS-polyacrylamide gel electrophoresis,
transferred to PVDF membranes, and immunoblotted with anti-HA antibody.
The film was exposed at room temperature for 30 s. B,
replicate membranes of protease protection products, generated as
described above, were immunoblotted with an anti-PEMT antibody.
C, replicate membranes of protease protection products,
generated as described above, were immunoblotted with an anti-PDI
antibody to confirm the integrity of the microsomes. Representative
immunoblots are shown. Each protease protection experiment was repeated
at least three times with similar results. D, predicted
membrane topography model of PEMT. Endoproteinase Lys-C cleavage sites
are denoted by arrows, and the length of cleavage fragments
generated from proteolysis at each site, as measured from the
N-terminal HA-tagged epitope, are indicated in kDa.
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Fig. 5.
Epitope-tagged mutant PEMT derivatives retain
enzymatic activity in transfected COS-7 cells. COS-7 cells, set up
as described under "Experimental Procedures," were transiently
transfected with 3 µg of plasmids containing epitope-tagged PEMT or
epitope-tagged mutant PEMT derivatives, or they were mock transfected
with empty pCI vector. A, cellular homogenates, 50-µg
protein, were assayed for PEMT activity. The results are expressed as
the mean of three separate experiments, each performed in duplicate, ± S.E., relative to the values obtained for similar assays on cells
transfected with HA-tagged PEMT. B, cellular homogenates,
25-µg protein, were immunoblotted with anti-HA antibody.
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Fig. 6.
Protease protection analysis of
the PEMT mutant CK2R2 confirms the cytosolic orientation of the C
terminus. A, predicted membrane topography model of
PEMT. Endoproteinase Lys-C cleavage sites are denoted by
arrows, and the length of cleavage fragments generated from
proteolysis at each site, as measured from the N-terminal HA-tagged
epitope, are indicated in kDa. Ablation of the C-terminal
endoproteinase cleavage sites is indicated. B, microsomes
were prepared from transfected cells as described under "Experimental
Procedures." Aliquots, 50-µg protein, were incubated with various
concentrations of endoproteinase at 37 °C for 3 h, in the
absence or presence of 1% Triton X-100. Reactions were stopped by the
addition of electrophoresis loading buffer and boiling at 100 °C for
10 min. Samples were separated by SDS-polyacrylamide gel
electrophoresis, transferred to PVDF membranes, and immunoblotted with
anti-HA antibody. The film was exposed at room temperature for 30 s. C, duplicate membranes of protease protection products,
generated as described above, were immunoblotted with an anti-PDI
antibody to confirm the integrity of the microsomes. Representative
immunoblots are shown. Each protease protection experiment was repeated
at least three times with similar results.
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Fig. 7.
Luminal orientation of loops A and C
demonstrated by protease protection analysis of PEMT mutant,
AK2R2. A, predicted membrane topography model of PEMT.
Endoproteinase Lys-C cleavage sites are denoted by arrows,
and the length of cleavage fragments generated from proteolysis at each
site, as measured from the N-terminal HA-tagged epitope, are indicated
in kDa. Ablation of the loop A endoproteinase cleavage sites is
indicated. B, microsomes were prepared from transfected
cells as described under "Experimental Procedures." Aliquots,
50-µg protein, were incubated with various concentrations of
endoproteinase at 37 °C for 3 h, in the absence or presence of
1% Triton X-100. Reactions were stopped by the addition of
electrophoresis loading buffer and boiling at 100 °C for 10 min.
Samples were separated by SDS-polyacrylamide gel electrophoresis,
transferred to PVDF membranes, and immunoblotted with anti-HA antibody.
The film was exposed at room temperature for 30 s. C,
duplicate membranes of protease protection products, generated as
described above, were immunoblotted with an anti-PDI antibody to
confirm the integrity of the microsomes. Representative immunoblots are
shown. Each protease protection experiment was repeated at least three
times with similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical regions are short hydrophilic loops (A, B, and C) that
range from 8 to 29 residues in length. Although the exact functional
significance of the length of the short loops remains undefined, this
structural organization may facilitate juxtaposition of distinct
functional domains from the adjoining transmembrane
-helices. Such
an alignment is not without precedent, as the topography of two enzymes
central to cellular cholesterol homeostasis (i.e. sterol
regulatory element-binding protein cleavage-activating protein and
3-hydroxy-3-methylglutaryl CoA reductase) features a series of five
closely aligned transmembrane domains that together constitute a
conserved sterol-sensing domain (35, 36).
-helical regions are not predicted to reorient within the membrane plane.
-helices. Elucidation of the topographical
organization of PEMT should therefore accelerate the identification of
residues that are important for binding AdoMet.
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ACKNOWLEDGEMENTS |
---|
We thank Susanne Lingrell for invaluable technical assistance. We acknowledge Drs. Belinda Hsi and Norm Kneteman for human liver samples and Wen-Hui Gao for microsome samples during initial topography studies. We thank Jenny Altarejos for production of the graphical topography models.
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FOOTNOTES |
---|
* This research was supported by a grant from the Canadian Institutes for Health Research. Ethics approval for work on human tissues was obtained from the Health Research Ethics board at the University of Alberta.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.
Supported by a Studentship from the Alberta Heritage Foundation
for Medical Research.
§ Scholar of the Alberta Heritage Foundation for Medical Research.
¶ Senior Scholar of the Alberta Heritage Foundation for Medical Research.
Canada Research Chair in Molecular and Cell Biology of Lipids
and Heritage Medical Scientist of the Alberta Heritage Foundation for
Medical Research. To whom correspondence should be addressed: 328 HMRC, Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-8286; Fax:
780-492-3383; E-mail: Dennis.Vance@ualberta.ca.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M210904200
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ABBREVIATIONS |
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The abbreviations used are: PC, phosphatidylcholine; AdoMet, S-adenosylmethionine; ER, endoplasmic reticulum; MAM, mitochondria-associated membranes; PDI, protein disulfide isomerase; PEMT, phosphatidylethanolamine N-methyltransferase; h, human; VLDL, very low density lipoprotein; HA, hemagglutinin; PVDF, polyvinylidene difluoride.
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