From the Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
Received for publication, September 19, 2002, and in revised form, November 26, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serine palmitoyltransferase (SPT), the
enzyme catalyzing the initial step in the biosynthesis of
sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1
has a single highly hydrophobic domain near the N terminus. Chinese
hamster ovary cell mutant LY-B cells are defective in SPT activity
because of the lack of expression of an endogenous LCB1 subunit. Stable
expression of LCB1 having an epitope tag at either the N or C terminus
restored SPT activity of LY-B cells, suggesting that the epitope tag
did not affect the localization or topology of LCB1. Indirect
immunostaining showed that the N- and C-terminal epitopes are oriented
toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in
wild-type cells, and the amount of LCB2 in LY-B cells was restored to
the wild-type level by transfection with LCB1 cDNA. In
addition, overproduction of the LCB2 subunit required co-overproduction
of the LCB1 subunit. These results indicated that the LCB1 subunit is
most likely an integral protein having a single transmembrane domain
with a lumenal orientation of its N terminus in the endoplasmic
reticulum and that the LCB1 subunit is indispensable for the
maintenance of the LCB2 subunit in mammalian cells.
Serine palmitoyltransferase
(SPT)1 is the enzyme
catalyzing the initial step of sphingolipid biosynthesis and catalyzes
the condensation of palmitoyl coenzyme A (CoA) with
L-serine to generate 3-ketodihydrosphingosine (1). In
mammalian cells, SPT consists of two different subunits, LCB1 and LCB2,
both of which are required for the enzymic activity (2-5). It has
recently been revealed that several missense mutations in
SPTLC1, the human LCB1 gene, cause hereditary sensory
neuropathy type I (6, 7) and that these mutations confer
dominant-negative effects on SPT activity (8, 9), supporting the notion
that the LCB1 subunit plays a crucial role in the formation of an
active SPT.
Comparisons of deduced amino acid sequences showed that there is about
30% identity between the mammalian LCB1 and LCB2 subunits and that
both can be affiliated to a subfamily of pyridoxal phosphate (PLP)-dependent enzymes, which includes oxononanoate
synthase, amino levulinate synthase, and amino ketobutyrate CoA ligase
(2, 5, 10-12). The subfamily members including SPT catalyze similar reactions, the condensation of carboxyl CoA thioesters with amino acids
to generate Previous biochemical studies have shown that SPT is a membrane-bound
protein enriched in the endoplasmic reticulum (ER) (14, 15).
Consistently, hydrophobic profiles of deduced amino acid sequences
predict that both the LCB1 and LCB2 subunits contain, at least, single
transmembrane domain (TMD) motifs (2, 5, 6, 10-12). However, the
number of possible TMDs depends on algorithms used for prediction,
indicating the necessity of experimental approaches to determine the
topology of the SPT subunits.
In the present study, we determine the topology of the LCB1 subunit
experimentally. In addition, we show that the LCB1 subunit of mammalian
SPT is indispensable for the maintenance of the LCB2 subunit.
Materials--
Mouse monoclonal anti-FLAG M2 antibody,
anti- Plasmids--
pSV-cLCB1 and pSV-cLCB2 are mammalian expression
plasmids for hamster LCB1 and LCB2 cDNA,
respectively (2). pSV-FHcLCB1 encodes a hamster LCB1 protein tagged
with FLAG and hexahistidine sequences at the N terminus (4). To
introduce an N-terminal hemagglutinin epitope (HA) tag into pSV-cLCB1,
a PCR was performed with pSV-cLCB1 as the template, a synthetic
oligonucleotide
(5'-TTTAGTCGACCATGTACCCATATGACGTCCCGGACTACGCCATGGCGATGGCGGCGGAGC-3') as the forward primer, and an oligonucleotide
(5'-TGCTCTAGACTACAGCAGCACAGCCTGGGC-3') as the reverse primer. To
introduce a C-terminal HA tag into pSV-cLCB1, PCR was performed with
pSV-cLCB1 as the template, an oligonucleotide (5'-ATTTAGGTGACACTATAGAAGGTACG-3') as the forward primer, and an
oligonucleotide
(5'-TGCTCTAGACTAGGCGTAGTCCGGGACGTCATATGGGTACAGCAGCACAGCCTGGGC-3') as
the reverse primer. The SalI-XbaI fragments of
the PCR products were ligated to
SalI-XbaI-digested pSV-OK, and the constructs encoding N- and C-terminal HA-tagged cLCB1 were named pSV-HAcLCB1 and
pSV-cLCB1HA, respectively. The nucleotide sequence of the constructs
was verified using an automated DNA sequencer (ABI PRISMTM310; Applied Biosystems).
Cell Culture and Transfection--
Chinese hamster ovary (CHO)
cells were routinely maintained in Ham's F-12 medium supplemented with
10% newborn bovine serum and antibiotics at 33 °C as described
previously (3). The LY-B strain, a mutant derived from strain CHO-K1,
and its stable transformants (LY-B/cLCB1 and LY-B/FHcLCB1) were
established previously by us (3, 4). Transfection of CHO cells was
performed with LipofectAMINE PLUSTM reagent (Invitrogen), according to
the manufacturer's manual. For the analysis of transiently transfected
cells, cell lysate was prepared 48 h after the start of
transfection. If necessary, after selection with G418 (400 µg/ml),
stable transfectants were isolated by limiting dilution. Stable
transfectants were systematically named after the names of their
parental strains and cLCB1 constructs (i.e. CHO-K1/cLCB1,
LY-B/HAcLCB1, and LY-B/cLCB1HA).
Preparation of Cell Lysate and Membranes--
All manipulations
were performed at 4 °C or on ice. After being washed with
phosphate-buffered saline (PBS), cells were harvested by scraping and
precipitated by centrifugation (300 × g, 5 min). The
precipitated cells were suspended in 10 mM Hepes-NaOH
buffer (pH 7.5) containing 250 mM sucrose, 1 mM
EDTA, and a protease inhibitor mixture (Roche Molecular Biochemicals)
and were disrupted by sonication. If necessary, membranes were prepared
from the sonicated lysate as described previously (16). Protein
concentrations of the preparations were determined with the Pierce
bicinchoninic acid protein assay kit with bovine serum albumin (BSA) as
the standard.
Western Blotting--
Western blot analysis of the LCB1 and LCB2
subunits of CHO cells was performed using the rabbit anti-cLCB1 73/90
and anti-cLCB2 26/45 antibodies, respectively, with an enhanced
chemiluminescence system as described previously (3, 4). We routinely
prepared two separate blots, each of which was used for analysis of
each LCB subunit, because incomplete stripping of the anti-cLCB1
antibody interfered with reprobing analysis of LCB2. For reprobing to
detect Grp94 and Grp78 as loading controls, the membrane blots were
incubated in PBS supplemented with 0.1% Tween 20, 1 M NaCl, and 100 mM 2-mercaptoethanol at
25 °C for 1 h, blocked, and processed with the mouse anti-KDEL monoclonal antibody and a horseradish peroxidase-conjugated anti-mouse IgG antibody as the primary and secondary antibodies, respectively. For
Western blotting of the HA epitope, the rat monoclonal anti-HA antibody
and a horseradish peroxidase-conjugated anti-rat IgG antibody (Amersham
Biosciences) were used as the primary and secondary antibodies,
respectively. For quantitative comparisons, the chemiluminescence in
blot membranes was analyzed with a LAS-1000plus lumino image analyzer
(Fuji Film, Tokyo, Japan).
Assay of SPT Activity--
SPT activity was assayed as described
previously (17). In brief, membranes (100 µg) were incubated in 200 µl of 50 mM Hepes-NaOH buffer (pH 7.5) containing 5 mM dithiothreitol, 5 mM EDTA, 50 µM pyridoxal phosphate, 0.2 mM palmitoyl CoA,
and 0.1 mM L-[3H(G)]serine (1.85 gigabecquerels/mmol). After incubation at 37 °C for 10 min,
the amount of [3H]3-ketodihydrosphingosine formed was measured.
Immunofluorescence Staining--
All manipulations were
performed at room temperature unless noted otherwise. LY-B/cLCB1,
LY-B/HAcLCB1, and LY-B/cLCB1HA cells, grown on glass coverslips at
37 °C, were fixed with 3.7% formaldehyde in PBS for 20 min and then
incubated with 0.1 M NH4Cl in PBS for 20 min.
After a rinse with PBS twice, the cells were incubated with 5 µg/ml
of digitonin in PBS for 10 min on ice for selective permeabilization of
the plasma membrane or 0.2% Triton X-100 in PBS for 15 min for
permeabilization of intracellular membranes. After a wash with PBS for
5 min three times, the cells were blocked with 3% BSA in PBS for 30 min. The cells were incubated for 1 h with one or a combination of
the following primary antibodies: 1) anti-HA antibody (1 µg/ml), 2)
anti-KDEL antibody (5 µg/ml), or 3) anti- Northern Blotting--
Total RNA was prepared from CHO cells
with an RNA isolation kit (ISOGEN; Nippon Gene, Toyama, Japan). After
electrophoresis in an agarose gel, RNA was blotted onto a nylon
membrane (Hybond N; Amersham Biosciences). The membrane was hybridized
with a 32P-labeled fragment of the hamster LCB2
cDNA under stringent conditions, and hybridizing LCB2
mRNA was detected by autoradiography.
Co-immunoprecipitation of the LCB2 Subunit with a FLAG-tagged
LCB1 Subunit--
All manipulation were performed at 4 °C unless
noted otherwise. After transfection of CHO-K1 cells with various
plasmids, membranes were prepared from the cells as described above.
Membranes (0.2 mg of protein) were solubilized with 1% (w/v) sucrose
monolaurate in 400 µl of Buffer A (0.1 M sodium phosphate
buffer (pH 8.0) containing 0.3 M NaCl and 0.1 M
sucrose). After centrifugation (105 × g, 30 min), supernatant fluid (~400 µl) was collected as the fraction of
solubilized membranes. The solubilized membranes (300 µl) were
incubated with anti-FLAG M2 affinity gel (25 µl of bed volume)
equilibrated with Buffer A containing 0.1% sucrose monolaurate for
1 h. After precipitation of the resin (104 × g, 30 s), the supernatant was collected as the unbound
fraction. Pelleted resin was washed three times with 1 ml of Buffer A
containing 0.1% sucrose monolaurate. Then, the washed resin was
incubated in 300 µl of Buffer EL (Buffer A supplemented with 50 mM Tris-HCl (pH 6.8), 2% (w/v) sodium dodecyl sulfate,
10% (w/v) glycerol, 50 mM dithiothreitol, and 0.1%
sucrose monolaurate) at 70 °C for 5 min. After precipitation of the
resin, the supernatant was collected as the bound fraction. Collected
fractions were subjected to Western blot analysis of LCB1 and LCB2
subunits as described above.
Immunoprecipitation of HA-tagged Protein--
All manipulation
were performed at 4 °C unless noted otherwise. Membranes from
LY-B/cLCB1, LY-B/HAcLCB1, and LY-B/cLCB1HA cells were solubilized with
1% sucrose monolaurate as described above. The solubilized membrane
fraction (200 µl) was incubated with protein G-Sepharose 4 Fast Flow
(Amersham Biosciences) (25 µl of bed volume) complexed with anti-HA
antibody equilibrated with Buffer A containing 0.1% sucrose
monolaurate and 0.1% BSA for 3 h. After centrifugation of the
resin (104 × g, 30 s), the supernatant
was collected as the unbound fraction. The gel was washed with Buffer A
containing 0.1% sucrose monolaurate three times and then incubated in
200 µl of Buffer EL at 70 °C for 5 min. After precipitation of the
gel, the supernatant was collected as the bound fraction. The
solubilized membranes and unbound fraction were used for assay of SPT
activity and Western blotting. The bound fraction was used for the
Western blotting.
Hamster LCB1 Derivatives Having the HA Epitope at N and C Termini
Are Functional--
The hydropathy profile of the LCB1 sequence shows
that there is a single highly hydrophobic domain (HD1) near the
N-terminal region and several moderately hydrophobic domains in other
regions (Fig. 1A). Some
programs for predicting TMD assign HD1 as a single TMD in the LCB1
protein (1-TMD model), whereas other programs assign HD1 and HD3
as possible TMDs (2-TDM model), and still others assign all of HD1-HD4
as TMDs (4-TMD model) (Fig. 1B). To distinguish the 1-TMD
model from the others experimentally, we decided to examine the
orientation of the N and C termini of the LCB1 protein. For this,
HAcLCB1 and cLCB1HA, which are hamster LCB1 variants having an HA
epitope at the N and C terminus, respectively (Fig. 2A), were stably expressed in
LY-B cells.2 The LY-B cell
line is a CHO cell mutant defective in SPT activity because of a lack
of expression of the LCB1 subunit of SPT (3). For fear that extreme
overproduction of a membrane protein would affect its localization
and/or topology, we selected stable transfectant clones (referred to as
LY-B/HAcLCB1 and LY-B/cLCB1HA, respectively), in which HA-tagged LCB1
levels were ~5-fold or less of the endogenous LCB1 level observed in
wild-type CHO cells (Fig. 2B, upper panel), for
further analysis. Importantly, SPT activity was restored in these LY-B
transfectants to the wild-type level (Fig. 2C), indicating that both the HAcLCB1 and cLCB1HA constructs are functional.
LY-B/HAcLCB1 cells unexpectedly produced an N-terminally truncated form
of HAcLCB1 at a low level, along with the full-sized protein (Fig. 2A, lane 4 of upper versus
middle panels).3
Nevertheless, when solubilized membranes from LY-B/HAcLCB1 were subjected to immunoprecipitation with anti-HA antibodies, ~80% of
the HAcLCB1 protein, but not its N-terminally truncated form, was
immunoprecipitated (Fig. 3A,
upper panel). Under such conditions, ~60% of the LCB2
subunit was co-immunoprecipitated with the HAcLCB1 protein (Fig.
3A, lower panel), and ~60% of SPT activity
also disappeared from the nonimmunoprecipitable fraction (Fig.
3B). Neither the endogenous LCB2 subunit nor SPT activity in
LY-B/cLCB1 was immunoprecipitated with anti-HA antibodies, whereas most
of SPT activity and the endogenous LCB2 subunit in LY-B/cLCB1HA cells were co-immunoprecipitated with the cLCB1HA protein, confirming the
specificity of the immunoprecipitation (Fig. 3, A and
B). These results demonstrated that the full size of HAcLCB1
is functional.
We also observed that there was far lower LCB2 in LY-B cells than in
wild-type CHO cells and that the amount of the LCB2 subunit in LY-B
cells was restored to the wild-type level by stable transfection with
the cLCB1 or HA-tagged cLCB1 constructs (Fig. 2B,
lower panel, lane 2 versus other
lanes). The LCB1-dependent alteration of LCB2 will be
further analyzed later in this paper.
Topology of the LCB1 Subunit at the ER--
The orientation of the
HA-tagged N and C termini of the LCB1 protein was determined by
antibody accessibility in appropriately permeabilized cells. Digitonin
treatment of fixed cells selectively permeabilizes the plasma membrane,
whereas Triton X-100 treatment permeabilizes all intracellular
membranes (18, 19). Indeed, when immunostained with an antibody against
The immunofluorescence staining also enabled us to explore the
intracellular distribution of the HA-tagged LCB1 at the confocal microscopic level. When Triton X-100-treated LY-B/HAcLCB1 and LY-B/cLCB1HA cells were doubly immunostained with anti-KDEL and anti-HA
antibodies, both signals were distributed to the reticular structure
residing throughout the cytoplasm and largely co-localized (Fig.
5), demonstrating that the HA-tagged
LCB1 proteins predominantly reside in the ER. It might also be
noteworthy that, when compared with the signal distribution of Grp78
and/or Grp94, signal for the HA-tagged LCB1 proteins was denser in the
perinuclear region than in other regions of the cytoplasmic reticular
membranes (see green signal in Fig. 5, C and
F). Collectively, these results indicated that the
N-terminal HA epitope of the LCB1 subunit is oriented into the lumen of
the ER and that the C-terminal epitope is oriented toward the cytosol.
This experimentally determined orientation of the N and C termini of
the HA-tagged LCB1 proteins is compatible with the
Nin-Cout type of the 1-TMD model but not with
other models shown in Fig. 1B.
The Lack of the LCB1 Subunit Causes the Reduction in the Level of
LCB2 in CHO Cells--
To determine whether a lack of the LCB1 subunit
affected the level of LCB2, we examined the levels of both subunits in
various CHO cell strains by Western blotting. The level of LCB1 in LY-B cells was less than 10% of that in wild-type CHO-K1 cells (Fig. 6A, lane 1 versus
lane 2), consistent with our previous studies (3, 4).
Interestingly, the level of the LCB2 subunit in LY-B cells was only
~10% of the wild-type (Fig. 6A, lane 1 versus lane 2). LCB2 levels in LY-B cells were restored to the
wild-type level when cDNA of hamster LCB1 (cLCB1) or its
FLAG- and hexahistidine-tagged version (FHcLCB1) was
introduced into LY-B cells by stable transfection (Fig. 6A,
lanes 3 and 4). We also introduced
cLCB1 to CHO-K1 cells and obtained a stable transformant
named CHO-K1/cLCB1. Although LCB1 levels in LY-B/cLCB1 and CHO-K1/cLCB1
cells were ~5-fold the wild-type level, such overproduction of LCB1
did not increase the amount of LCB2 beyond the wild-type level (Fig.
6A). Reprobing of the blots with the anti-KDEL antibody
showed that the levels of Grp94 and Grp78 were virtually equal among
the cell types examined, eliminating the possibility that the reduced
levels of both LCB1 and LCB2 in LY-B cells were because of nonspecific
protein degradation. Similar results were observed when LY-B cells were
transfected with HA-tagged cLCB1 constructs (Fig. 2B). It
should also be noted that there was no difference in the
LCB2 mRNA level between CHO-K1 and LY-B cells (Fig.
6B). These results demonstrated that the lack of LCB1
reduces the steady state level of LCB2 in CHO cells, suggesting that,
when LCB1 is missing, LCB2 can not exist stably in mammalian cells.
Overproduction of the LCB2 Subunit Requires Co-overproduction of
the LCB1 Subunit--
To further examine whether the LCB1 subunit
played a crucial role in the maintenance of the LCB2 subunit, we
analyzed the levels of the two after the transient transfection of
CHO-K1 cells with various plasmids. Mock transfection with the empty
pSV vector affected neither LCB1 nor LCB2 (Fig.
7, lane 1 versus lane
2). On transfection with pSV-cLCB1, the LCB1 subunit was
overproduced to ~50-fold or more of the wild-type level regardless of
co-transfection with pSV-cLCB2 (Fig. 7, lane 1 versus
lanes 3 and 5). In contrast, transfection with
pSV-cLCB2 without pSV-cLCB1 caused only an ~2-fold increase in LCB2,
compared with the non-transfected control (Fig. 7, lane 1 versus lane 4). However, when cells were co-transfected with pSV-cLCB1 and pSV-cLCB2, the level of overproduction of the LCB2
subunit became comparable with that of the LCB1 subunit (Fig. 7,
lane 5). Reprobing of the blots with the anti-KDEL antibody confirmed loading of equal protein amount. These results indicated that
overproduction of the LCB2 subunit requires co-overproduction of the
LCB1 subunit.
To examine whether the overproduced LCB2 subunit was associated with
the LCB1 subunit, we carried out co-immunoprecipitation experiments
using a FLAG epitope-tagged LCB1. Co-transfection of CHO-K1 cells with
pSV-cLCB2 and pSV-FHcLCB1 resulted in overproduction of the LCB2
subunit (Fig. 8, lane 2 versus
lane 4), similar to the case of co-transfection with
pSV-cLCB2 and pSV-cLCB1 (see Fig. 7 and Fig. 8, lane 3).
When the solubilized membrane fraction of the cells was incubated with
an anti-FLAG antibody-coupled resin, most of the overproduced LCB2 was
co-immunoprecipitated with the FLAG-tagged LCB1 protein (Fig. 8,
lane 12). The possibility of nonspecific binding of the LCB2
subunit to the resin was eliminated, because, when cells
were transfected with pSV-cLCB1 in place of pSV-FHcLCB1, no LCB2 was
immunoprecipitated (Fig. 8, lane 11). In addition,
determination of protein concentrations showed that more than 95% of
proteins of the solubilized membrane fraction were distributed to the
nonimmunoprecipitable fraction (data not shown), ruling out the minor
possibility that the membranes had not been completely solubilized, so
that anti-FLAG antibody precipitated all proteins of the
FLAG-LCB1-expressing membranes. These results demonstrated that almost
all of the overproduced LCB2 subunit forms a complex with the
overproduced LCB1 subunit. It should also be pointed out that the
endogenous non-tagged LCB1 protein was not co-immunoprecipitated with
the FLAG-tagged LCB1 protein (Fig. 8, top panel, lanes
6 and 8 versus lanes 10 and 12),
indicating that the LCB1 protein does not form any homo-oligomers.
Although hydrophobic profiles of the primary structure of
membrane-bound proteins predict possible TMDs and also their membrane topology, different algorithms sometimes give different predictions (21, 22). Therefore, for the elucidation of topology of membrane proteins, experimental approaches remain indispensable. In the present
study, we determined the orientation of the N and C termini of the
hamster LCB1 subunit by using epitope-tagged constructs (Fig. 4) and
also showed the predominant distribution of this protein in the ER at
the confocal microscopic level (Fig. 5). The ER localization of the
LCB1 subunit is consistent with previous studies showing that SPT
activity is enriched in the ER fraction among various
subcellular fractions (14, 15). From the hydropathy profile
of the LCB1 protein, together with our experimental results shown
above, we conclude that hamster LCB1 is an integral protein having a
single TMD with a lumenal orientation of its N terminus in the ER. One
might imagine other possible models, in which the protein had three or
five TDMs, because LCB1 has several moderately hydrophobic regions
(Fig. 1A). However, such regions (including HD2-4 in Fig. 1A) show significant
similarities to corresponding regions of soluble enzymes of the POAS
family in sequence alignments. In contrast, the highly hydrophobic
domain of the LCB1 protein (HD1 in Fig. 1A) has
no similarity to any other member of the family. These bioinformatic
analyses strongly suggest the moderately hydrophobic regions of the
LCB1 protein serve as internal domains of a globular part of the
protein, not as TMDs, although further studies would be needed to prove
this prediction.
The topological assignment we made above is most likely valid in LCB1
proteins of other organisms, because hamster LCB1 has ~95% identity
to human and mouse LCB1 and 35% identity to yeast Lcb1p at the amino
acid level (2, 5). From the topological model for the LCB1 subunit, the
catalytic site of SPT can be deduced to be oriented to the cytosolic
space at the ER, assuming that the catalytic site of mammalian SPT is
formed at the interface of the LCB1 and LCB2 subunits as discussed
below. This deduction is consistent with a previous study (15) showing
that SPT bound to isolated intact microsomes is inactivated by
externally added proteases. The cytosolic orientation of the catalytic
site of SPT is probably relevant to the accessibility of enzyme
substrates, because the cytosol is the major pool of serine and
palmitoyl CoA in cells.
In the present study, we obtained evidence that the LCB1 subunit is
required for the maintenance of the LCB2 subunit in mammalian cells.
First, the amount of LCB2 in CHO mutant cells lacking the endogenous
LCB1 subunit was found to be far lower than the wild-type level (see
Fig. 2B and Fig. 6A). When the LCB1 subunit was
expressed in the mutant cells by transfection, the LCB2 level was
restored to the wild-type level (see Fig. 2B and Fig.
6A). In addition, overproduction of the LCB2 subunit in CHO
cells required overproduction of the LCB1 subunit (see Figs. 7 and 8).
The requirement of LCB1 expression for the maintenance of the LCB2
subunit was unlikely because of a possible transcriptional regulation
of the LCB2 gene by the level of the LCB1 subunit, because
the LCB2 mRNA level in LY-B cells is similar to that in
CHO-K1 cells (Fig. 6B). In addition,
LCB1-dependent alteration of the LCB2 subunit was observed even when the expression of cLCB1 and cLCB2 was driven by an
SV40-derived promoter (see Figs. 7 and 8). Probably,
post-transcriptional events cause the alteration of the LCB2 subunit.
Taking account of the fact that almost all of the overproduced LCB2
protein molecules are associated with the LCB1 subunit (Fig. 8), we
suggest that the LCB2 subunit is unstable unless it is associated with
the LCB1 subunit.
The nature of the LCB2 subunit, which requires the LCB1 subunit for
maintenance, may be conserved among eukaryotes, because a lack of Lcb1p
also causes a reduction in Lcb2p in the yeast Saccharomyces
cerevisiae (23). By contrast, the LCB1 subunit can be highly
overproduced without co-overproduction of the LCB2 subunit (see Figs. 7
and 8). These results suggest that, if a pool of free LCB1 subunit is
available, cells can regulate the level of LCB1/LCB2 complex through
transcriptional regulation of the LCB2 gene even without
parallel regulation of LCB1. This scenario might account for
the previously reported observation that the change in SPT activity in
response to ultraviolet irradiation parallels the change in the
LCB2 mRNA level but not the LCB1 mRNA level in cultured human keratinocytes (24). The nature of the LCB2
subunit could also serve to prevent harmful side reactions by free LCB2
having a latently reactive PLP. The LCB1 subunit has no PLP-binding
motif and, therefore, is theoretically inert. Thus, free LCB1 might not
be severely toxic.
We previously demonstrated that hamster LCB1 forms a complex with
hamster LCB2 at a molecular ratio of 1:1 (4). In the present study, we
further showed that, when expressed in wild-type CHO cells, a
FLAG-tagged LCB1 was co-immunoprecipitated with the endogenous (and
also co-overproduced) LCB2 but not any endogenous LCB1 (Fig. 8). These
results demonstrate that two LCB1 molecules are not simultaneously
integrated into one LCB1/LCB2 complex, consistent with the results
observed in a yeast system (9). Although additional gene product,
Tsc3p, is required for the optimum activity of SPT in yeast cells (23),
no mammalian homolog of Tsc3 has been found even in the
human genome data base. Moreover, no proteins except for LCB1 and LCB2
proteins were detected in silver staining patterns of purified hamster
SPT (4). Collectively, it is most likely that mammalian SPT is a
heterodimer of the LCB1 and LCB2 subunits, although as yet unknown
factors might associate with the LCB1/LCB2 complex to regulate the
activity of SPT in vivo.
Analysis of the tertiary structure of bacterial oxononanoate synthase,
a member of the POAS family, has revealed that the catalytic site forms
at the interface of each subunit of this homodimer enzyme (25, 26). By
analogy, SPT is suggested to form its catalytic site at the interface
of the subunits in the LCB1/LCB2 heterodimer. If so, the LCB1 subunit
may play crucial roles in the formation of the enzyme catalytic site,
besides the role in the maintenance of the LCB2 subunit. This is
supported by recent studies showing that several specific missense
mutations in LCB1 confer dominant negative effects on SPT
activity without affecting the steady state level of LCB2 subunit in
yeast (9) and mammalian cells (8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxoamines. Eukaryote SPT is a membrane-bound enzyme
consisting of different subunits, whereas other members of the
PLP-dependent
-oxoamine synthase (POAS) family are
soluble homodimers. Curiously, although the LCB2 subunit of SPT has the PLP-binding motif conserved in the POAS family, the LCB1 subunit does
not (2, 5, 10-12). In addition, the Gram-negative bacterium Sphingomonus paucimobilis produces a soluble SPT as a
homodimer of a protein having the PLP-binding motif (13). These results suggest that the LCB1 subunit has a function(s) specific for the heterosubunit type of SPT. However, it remains poorly understood why
mammalian SPT comprises an LCB1 subunit, as well as the LCB2 subunit,
having the PLP-binding motif.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin antibody (DM1A), and digitonin were purchased from
Sigma; rat monoclonal anti-HA antibody (3F10) was from Roche
Applied Science; mouse monoclonal anti-KDEL antibody (10C3) was
from StressGen (Victoria, British Columbia, Canada); Alexa
Fluor 488 and 594 anti-mouse IgG and Alexa Fluor 488 anti-rat IgG were
from Molecular Probes (Eugene, OR). Sucrose monolaurate was from
Dojindo (Kumamoto, Japan). L-[3H(G)]serine
(7.4 terabecquerels/mmol) was from Moravek Biochemicals (Brea, CA).
-tubulin antibody
(1:500). After a wash with PBS for 5 min three times, the cells were
incubated for 30 min with secondary antibody conjugated with Alexa
Fluor 488 and/or 594 (1:125) and washed with PBS for 5 min three times.
The specimens were observed with a fluorescent microscope (Axiovert
S100TV; Carl Zeiss, Tokyo, Japan) equipped with a digital charged
coupled device camera (model C4742-95-12; Hamamatsu Photonics,
Hamamatsu, Japan), and pictures were taken and viewed with IP Lab 3.21J
software (Scanalytics, Inc., Fairfax, VA). For analysis of the
intracellular localization of protein, the specimens were viewed with a
confocal laser scanning microscope (Axiovert 100M; Carl Zeiss)
equipped with a LSM510 system (Carl Zeiss).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Hydropathy plot and predicted membrane
topology of hamster LCB1. A, the hydropathy plot of
hamster LCB1 was generated by the method of Kyte and Doolittle (27)
using a window of 19 amino acids. B, different models of
membrane topology predicted by different programs. The hydropathy (27),
ALOM (28), and DAS (29) programs assign HD1 as a single transmembrane
domain (1-TMD Model). The TMHMM program (30) assigns HD1 and
HD3 as possible transmembrane domains (2-TMD Model). The
TMpred (31) and PHDhtm (32) programs assign HD1, HD2, HD3, and HD4 as
transmembrane domains (4-TMD Model). These programs are
available from public servers as listed in a review (21).
View larger version (19K):
[in a new window]
Fig. 2.
HAcLCB1 and cLCB1HA functionally substitute
for wild-type cLCB1 in CHO cells. A, N- and C-terminal
sequences of the cLCB1, HAcLCB1, and cLCB1HA products are shown.
B, membranes prepared from CHO-K1 (lane 1), LY-B
(lane 2), LY-B/cLCB1 (lane 3), LY-B/HAcLCB1
(lane 4), and LY-B/cLCB1HA (lane 5) cells were
analyzed by Western blotting with anti-hamster LCB1, anti-HA, and
anti-hamster LCB2 antibodies. C, SPT activity in membranes
from various cell lines indicated above was determined. The data are
shown as means ± S.D. from triplicate experiments.
View larger version (22K):
[in a new window]
Fig. 3.
Co-immunoprecipitation of LCB2 subunit and
SPT activity with HAcLCB1 and cLCB1HA. Solubilized membranes
(Sol. M.) were incubated with anti-HA antibody bound to
protein G-Sepharose as described under "Experimental Procedures."
After centrifugation of the resin, the supernatant was collected as
unbound fraction. The resin was washed with a wash buffer and incubated
in an SDS-sample buffer. After precipitation of the resin, the
supernatant was collected as the bound fraction. A, Western
blotting of the solubilized membranes and unbound and bound fractions.
Lanes 1, 4, and 7, samples from
LY-B/cLCB1 cells; lanes 2, 5, and 8,
samples from LY-B/HAcLCB1cells; lanes 3, 6, and
9, samples from LY-B/cLCB1HA cells. The faint
band (marked by asterisk) reacted with anti-LCB2
antibody might represent a modified form of LCB2, although identity of
the faint band remains unclear. B,
nonimmunoprecipitable SPT activity. SPT activity in the unbound
fraction is represented as the percentage of the activity in the
solubilized membranes. The data are shown as means ± S.D. from
triplicate experiments.
-tubulin, a cytosolic protein, signal was observed in
digitonin-treated cells, as well as in Triton X-100-treated cells (Fig.
4, C, F,
I, and L). In contrast, immunostaining of Grp78
(or BiP) and Grp94, both of which reside in the ER lumen (20), with
anti-KDEL antibodies was observed in Triton X-100-treated cells but not
in digitonin-treated cells (Fig. 4, B, E,
H, and K). Signal for the HA epitope in
LY-B/HAcLCB1 cells was detected after Triton X-100, but not digitonin,
treatment (Fig. 4, A versus D),
whereas signal in LY-B/cLCB1HA was observed under both conditions
almost equally (Fig. 4, G and J). No signal was
observed in LY-B/cLCB1 cells expressing non-tagged cLCB1 (Fig. 4M), indicating the specificity of the immunostaining for
the HA epitope.
View larger version (68K):
[in a new window]
Fig. 4.
The orientation of the HA-tagged N and C
termini of cLCB1 protein in appropriately permeabilized cells.
LY-B/HAcLCB1 (A-F), LY-B/cLCB1HA
(G-L), and LY-B/cLCB1 (M) cells grown
on glass coverslips were fixed and treated with 5 µg/ml of digitonin
(A-C and G-I) or 0.2%
Triton X-100 (D-F and
J-M). The cells were immunostained with anti-HA
(A, D, G, J, and
M), anti-KDEL (B, E, H, and
K), or anti- -tubulin (C, F,
I, and L) primary antibody and then with Alexa
Fluor-coupled secondary antibodies. The immunostained cells were
observed by fluorescence microscopy. Scale bar, 10 µm.
View larger version (35K):
[in a new window]
Fig. 5.
Intracellular distribution of HA-tagged cLCB1
proteins. The LY-B/HAcLCB1 (A-C) and
LY-B/cLCB1HA (D-F) cells, which had
been fixed and treated with Triton X-100, were immunostained with rat
anti-HA and mouse anti-KDEL primary antibodies and then with anti-rat
and anti-mouse IgG antibodies, coupled with Alexa Fluor 488 and 594, respectively. The immunostained cells were observed by confocal
microscopy. The localization of HA-tagged cLCB1 is shown in
green (A and D); the localization of
Grp78 and/or Grp94, both of which are ER proteins, is shown in
red (B and E); and co-localization
with green and red signals is shown in
yellow in the merged image (C and F).
Scale bar, 10 µm.
View larger version (58K):
[in a new window]
Fig. 6.
Reduction of the LCB2 subunit level because
of a lack of LCB1 subunit. A, the levels of LCB1 and
LCB2 subunits in the membrane fraction (20 µg of protein per well) of
various CHO cell lines were examined by Western blotting. Then, the
blots used for analysis of LCB proteins were reprobed with the
anti-KDEL antibody to examine the levels of Grp94 and Grp78 as loading
controls. The reprobed pattern is presented below the
pattern of each LCB blot. Lane 1, CHO-K1; lane 2,
LY-B; lane 3, LY-B/cLCB1; lane 4, LY-B/FHcLCB1;
lane 5, CHO-K1/cLCB1. B, total RNA fraction (25 µg/lane) prepared from CHO-K1 (lane 1) and LY-B
(lane 2) was subjected to Northern blot analysis of
LCB2 mRNA. The ~2.0-kb major hybrid corresponds to the
predominant form of LCB2 mRNA (2). The minor hybrid,
marked by an asterisk, is probably an alternative form of
LCB2 mRNA, although its identity remains unclear.
View larger version (48K):
[in a new window]
Fig. 7.
Overproduction of the LCB2 subunit requires
co-overproduction of the LCB1 subunit. Subconfluent CHO-K1 cells
grown in 35-mm dishes were transfected with pSV-cLCB1, pSV-cLCB2,
and/or the empty vector in the indicated combinations. Lysate (7.5 µg
of protein per well) prepared from the transfected cells was subjected
to Western blot analysis of LCB1 and LCB2 subunits. Then, the blots
used for analysis of the LCB proteins were reprobed with the anti-KDEL
antibody to examine the levels of Grp94 and Grp78 as loading controls.
The reprobed pattern is presented below the pattern of each
LCB blot.
View larger version (22K):
[in a new window]
Fig. 8.
Co-immunoprecipitation of the LCB2 subunit
with a FLAG-tagged LCB1 subunit. CHO-K1 cells were transfected,
and membranes were prepared from the cells. After solubilization with
1% sucrose monolaurate, the solubilized membrane fraction was
incubated with an anti-FLAG antibody-coupled resin, and then fractions
unbound and bound to the resin were obtained as described under
"Experimental Procedures." Equivalent volume of these fractions was
loaded into each lane and analyzed by Western blotting for
LCB1 and LCB2 subunits. Lane 1, 5, and
9, transfected with pSV-cLCB1 alone; lane 2,
6, and 10, transfected with pSV-FHcLCB1 alone;
lane 3, 7, and 11, co-transfected with
pSV-cLCB1 and pSV-cLCB2; lane 4, 8, and
12, co-transfected with pSV-FHcLCB1 and pSV-cLCB2.
Sol. M., solubilized membrane fraction; Unbnd.,
unbound fraction; Bound, bound fraction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants-in-aid KAKENHI 12140206 and 12680610 from the Ministry of Education, Culture, Sports, Science and Technology.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.
To whom correspondence should be addressed. Tel.: 81-3-5285-1111 (ext. 2126); Fax: 81-3-5285-1157; E-mail: hanak@nih.go.jp.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M209602200
2 FLAG-tagged cLCB1 constructs could not be used for cytochemical analysis, because substantial non-specific signal was detected in immunostaining of LY-B/cLCB1 cells with the anti-FLAG M2 antibody.
3 The N-terminally truncated form is probably because of translation from the second ATG codon in the HAcLCB1 construct, because the nucleotide sequence around the second ATG, rather than the first ATG, in the HAcLCB1 construct matches the Kozak consensus for efficient translational initiation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SPT, serine
palmitoyltransferase;
CoA, coenzyme A;
ER, endoplasmic reticulum;
CHO, Chinese hamster ovary;
PLP, pyridoxal phosphate;
POAS, PLP-dependent -oxoamine synthase;
TMD, transmembrane
domain;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Merrill, A. H., Jr.
(2002)
J. Biol. Chem.
277,
25843-25846 |
2. |
Hanada, K.,
Hara, T.,
Nishijima, M.,
Kuge, O.,
Dickson, R. C.,
and Nagiec, M. M.
(1997)
J. Biol. Chem.
272,
32108-32114 |
3. |
Hanada, K.,
Hara, T.,
Fukasawa, M.,
Yamaji, A.,
Umeda, M.,
and Nishijima, M.
(1998)
J. Biol. Chem.
273,
33787-33794 |
4. |
Hanada, K.,
Hara, T.,
and Nishijima, M.
(2000)
J. Biol. Chem.
275,
8409-8415 |
5. | Weiss, B., and Stoffel, W. (1997) Eur. J. Biochem. 249, 239-247[Abstract] |
6. | Bejaoui, K., Wu, C., Scheffler, M. D., Haan, G., Ashby, P., Wu, L., de Jong, P., and Brown, R. H., Jr. (2001) Nat. Genet. 27, 261-262[CrossRef][Medline] [Order article via Infotrieve] |
7. | Dawkins, J. L., Hulme, D. J., Brahmbhatt, S. B., Auer-Grumbach, M., and Nicholson, G. A. (2001) Nat. Genet. 27, 309-312[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Bejaoui, K.,
Uchida, Y.,
Yasuda, S., Ho, M.,
Nishijima, M.,
Brown, R. H., Jr.,
Holleran, W. M.,
and Hanada, K.
(2002)
J. Clin. Invest.
110,
1301-1308 |
9. |
Gable, K.,
Han, G.,
Monaghan, E.,
Bacikova, D.,
Natarajan, M.,
Williams, R.,
and Dunn, T. M.
(2002)
J. Biol. Chem.
277,
10194-10200 |
10. | Buede, R., Rinker-Schaffer, C., Pinto, W. J., Lester, R. L., and Dickson, R. C. (1991) J. Bacteriol. 173, 4325-4332[Medline] [Order article via Infotrieve] |
11. | Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L., and Dickson, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7899-7902[Abstract] |
12. |
Zhao, C.,
Beeler, T.,
and Dunn, T.
(1994)
J. Biol. Chem.
269,
21480-21488 |
13. |
Ikushiro, H.,
Hayashi, H.,
and Kagamiyama, H.
(2001)
J. Biol. Chem.
276,
18249-18256 |
14. | Williams, R. D., Wang, E., and Merrill, A. H., Jr. (1984) Arch. Biochem. Biophys. 228, 282-291[Medline] [Order article via Infotrieve] |
15. |
Mandon, E. C.,
Ehses, I.,
Rother, J.,
van Echten, G.,
and Sandhoff, K.
(1992)
J. Biol. Chem.
267,
11144-11148 |
16. |
Hanada, K.,
Nishijima, M.,
Akamatsu, Y.,
and Pagano, R. E.
(1995)
J. Biol. Chem.
270,
6254-6260 |
17. | Merrill, A. H., Jr. (1983) Biochim. Biophys. Acta 754, 284-291[Medline] [Order article via Infotrieve] |
18. | Roitelman, J., Olender, E. H., Bar-Nun, S., Dunn, W. A., Jr., and Simoni, R. D. (1992) J. Cell Biol. 117, 959-973[Abstract] |
19. |
Lin, S.,
Cheng, D.,
Liu, M.,
Chen, J.,
and Ta-Yuan Chang, T.
(1999)
J. Biol. Chem.
274,
23276-23285 |
20. | Munro, S., and Pelham, H. R. (1987) Cell 48, 899-907[Medline] [Order article via Infotrieve] |
21. | Chen, C. P., and Rost, B. (2002) Appl. Bioinform. 1, 21-35 |
22. |
Ott, C. M.,
and Lingappa, V. R.
(2002)
J. Cell Sci.
115,
2003-2009 |
23. |
Gable, K.,
Slife, H.,
Bacikova, D.,
Monaghan, E.,
and Dunn, T. M.
(2000)
J. Biol. Chem.
275,
7597-7603 |
24. |
Farrell, A. M.,
Uchida, Y.,
Nagiec, M. M.,
Harris, I. R.,
Dickson, R. C.,
Elias, P. M.,
and Holleran, W. M.
(1998)
J. Lipid Res.
39,
2031-2038 |
25. | Alexeev, D., Alexeeva, M., Baxter, R. L., Campopiano, D. J., Webster, S. P., and Sawyer, L. (1998) J. Mol. Biol. 284, 401-419[CrossRef][Medline] [Order article via Infotrieve] |
26. | Webster, S. P., Alexeev, D., Campopiano, D. J., Watt, R. M., Alexeeva, M., Sawyer, L., and Baxter, R. L. (2000) Biochemistry 39, 516-528[CrossRef][Medline] [Order article via Infotrieve] |
27. | Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve] |
28. | Klein, P., Kanehisa, M., and DeLisi, C. (1985) Biochim. Biophys. Acta 815, 468-476[Medline] [Order article via Infotrieve] |
29. | Cserzo, M., Wallin, E., Simon, I., von Heijne, G., and Elofsson, A. (1997) Protein Eng. 10, 673-676[Abstract] |
30. | Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. L. (2001) J. Mol. Biol. 305, 567-580[CrossRef][Medline] [Order article via Infotrieve] |
31. | Hofmann, K., and Stoffel, W. (1993) Biol. Chem. Hoppe-Seyler 374, 166 |
32. | Rost, B. (1996) Methods Enzymol. 266, 525-539[CrossRef][Medline] [Order article via Infotrieve] |