The Import of S-Adenosylmethionine into the Golgi
Apparatus Is Required for the Methylation
of Homogalacturonan1[W][OA]
Consuelo Ibar and Ariel Orellana*
Millennium Nucleus in Plant Cell Biology, Center of Plant Biotechnology, Andrés Bello University,
República 217, Santiago, Chile
S-adenosylmethionine (SAM) is the substrate used in the methylation of homogalacturonan (HGA) in the Golgi apparatus. SAM
is synthesized in the cytosol, but it is not currently known how it is then transported into the Golgi. In this study, we find that HGA
methyltransferase is present in Golgi-enriched fractions and that its catalytic domain faces the lumen of this organelle. This
suggests that SAM must be imported into the Golgi. We performed uptake experiments using [methyl-14C]SAM and found that
SAM is incorporated into the Golgi vesicles, resulting in the methylation of polymers that are sensitive to pectinase and pectin
methylesterase but not to proteases. To avoid detecting the transfer reaction, we also used [carboxyl-14C]SAM, the uptake of which
into Golgi vesicles was found to be sensitive to temperature, detergents, and osmotic changes, and to be saturable with a Km of 33
mM. Double-label uptake experiments using [methyl-3H]SAM and [carboxyl-14C]SAM also revealed a time-dependent increase
in the 3H to 14C ratio, suggesting that upon transfer of the methyl group, the resulting S-adenosylhomocysteine is not accumulated
in the Golgi. SAM incorporation was also found to be inhibited by S-adenosylhomocysteine, whereas UDP-GalA, UDP-GlcA, and
acetyl-CoA had no effect. DIDS, a compound that inhibits nucleotide sugar transporters, also had little effect upon SAM incorporation. Interestingly, the combination of UDP-GalA 1 acetyl-CoA or UDP-GlcA 1 acetyl-CoA produced a slight increase in
the uptake of SAM. These results support the idea that a SAM transporter is required for HGA biosynthesis.
S-adenosylmethionine (SAM) is the donor substrate
for a number of methylation reactions within the cell,
including DNA and protein methylation (Fontecave
et al., 2004). In plants, SAM is also involved in other
metabolic pathways, such as ethylene biosynthesis
(Kende, 1993) and the methylation of pectins (Kauss
and Hassid, 1967; Vannier et al., 1992; Boulard et al.,
1997; Goubet et al., 1998). The pectins are a group of
complex polysaccharides present in the primary cell
wall in plants and are composed of homogalacturonan
(HGA), rhamnogalacturonan I, and rhamnogalacturonan II (O’Neill et al., 1990; Albersheim et al., 1996).
Previous studies using monoclonal antibodies against
pectins and also involving immunoelectronmicroscopy
have suggested that the pectins are synthesized in the
Golgi apparatus (Zhang and Staehelin, 1992). In addition, Goubet and Mohnen (1999a) have described a
Golgi-localized HGA methyltransferase (HGA-MT) in
tobacco (Nicotiana tabacum) cells, thus indicating that
the methylation of HGA also takes place in the Golgi.
1
This work was supported by Fondecyt 1030551 and the Millennium Nucleus in Plant Cell Biology (ICM P 02–009–F).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to
the findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Ariel Orellana ([email protected]).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.104679
504
Furthermore, they also found that by treating Golgi
fractions with proteinase K, the HGA-MT activity was
decreased only in permeabilized membranes, suggesting that this enzyme is located in the lumen of the
Golgi apparatus.
The reported evidence to date indicates that SAM
biosynthesis occurs in the cytosol (Wallsgrove et al.,
1983; Ravanel et al., 1998). However, since HGA-MT
utilizes SAM in the lumen of the Golgi apparatus, this
substrate must necessarily be imported into this subcellular compartment. However, to our knowledge no
evidence for the transport of SAM into the Golgi apparatus has so far been provided. Hence, to establish
whether SAM is indeed transported into the Golgi and
used in the methylation of pectins, we have analyzed
the incorporation of SAM into Golgi vesicles obtained
from etiolated pea (Pisum sativum) epicotyls in this study.
Our results suggest that SAM is indeed transported
into the lumen of the Golgi and used in the methylation of pectins. Moreover, this importation process
shows features that differ from other Golgi-localized
transporters, such as the nucleotide sugar transporters
(NSTs), suggesting that a new class of Golgi transporter is involved in polysaccharide biosynthesis.
RESULTS
To determine the mechanisms underlying the importation of SAM into the Golgi, we purified Golgi
vesicles from etiolated pea epicotyls, an experimental
model plant that has been utilized previously to analyze
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Import of S-Adenosylmethionine into the Golgi
Figure 1. HGA-MT activity is found in the lumen of Golgi vesicles. A,
HGA-MT activity was measured for 1 h on intact or permeabilized
Golgi vesicles (50 mg of protein) in the presence (1) or absence (2) of
50 mg of HGA. Vesicles were treated with or without 0.1% (v/v) Triton
X-100. B, XG-FT was measured for 30 min on intact or permeabilized
Golgi vesicles (100 mg of protein) in the presence (1) or absence (2) of
100 mg of tamarind XG. Vesicles were permeabilized using 0.05% (v/v)
Triton X-100. C and D, Golgi vesicles (50 mg of protein) were treated for
10 min with 5 or 25 mg of trypsin, in the absence (see C) or presence
(see D) of 0.02% (v/v) Triton X-100. Trypsin was inactivated using
trypsin inhibitor as described in ‘‘Materials and Methods.’’ The trypsintreated vesicles were then used to assess the HGA-MT activity. The total
activity present in each sample was measured in the presence of HGA
(50 mg), [methyl-14C]SAM, and 0.1% (v/v) Triton X-100 as described in
‘‘Materials and Methods.’’ HGA was precipitated in 20% TCA and the
radioactivity associated to the pellet was measured using a liquid
scintillation counter.
the topology of enzymes involved in the biosynthesis
of polysaccharides, as well as the transport of metabolites and ions (Muñoz et al., 1996; Neckelmann and
Orellana, 1998; Wulff et al., 2000; Sterling et al., 2001;
Ordenes et al., 2002). We first determined the orientation of the catalytic domain of HGA-MT in our Golgi
vesicle preparations by measuring its enzymatic activity in the presence of exogenous HGA as the acceptor for the methylation reaction. This polymer cannot
access the lumen of the Golgi vesicles unless the
integrity of the membrane is altered, and we thus measured HGA-MT activity both in the presence and absence of 0.1% Triton X-100.
A 6-fold increase in the activity of HGA-MT was observed when the Golgi vesicle membranes were permeabilized with detergent (Fig. 1A). This activation level
was also similar to those observed for xyloglucan (XG)fucosyltransferase (FT) in the presence of 0.1% Triton
X-100 (Fig. 1B). XG-FT is a type II membrane protein
with a catalytic site that faces the lumen of the Golgi
apparatus (Perrin et al., 1999; Wulff et al., 2000). Since
HGA-MT and XG-FT were measured in the presence of
acceptors that cannot cross the membrane, the increases
we detected in their activity following the permeabilization of the Golgi vesicle membranes could be explained by the luminal localization of the active site of
both enzymes. To confirm whether the active site of
HGA-MT indeed faces the lumen of the Golgi, we
treated both intact and permeabilized Golgi vesicles
with trypsin to disrupt both the cytosolic and lumenal
domains of the membrane proteins and also any Golgi
soluble proteins. Following these trypsin treatments,
the intact vesicles were also permeabilized and the
total remaining HGA-MT activity was measured using
exogenous HGA (Fig. 1, C and D). The results show
that trypsin has no effect on the HGA-MT activity in
intact vesicles, but almost completely abolishes this
activity in already Triton X-100-treated Golgi vesicles.
These results strongly suggest that the catalytic domain of HGA-MT faces the lumen of the Golgi apparatus
in etiolated peas. These results are also in agreement
with the previously reported findings of Goubet and
Mohnen (1999a) in tobacco cells.
Since the newly synthesized HGA and HGA-MT are
located in the lumen of the Golgi, we postulated that
SAM would also need to be incorporated into this compartment. To test this, we performed subcellular fractionation of etiolated pea stem organelles and measured
the uptake of SAM into these different subcellular fractions. We observed that the uptake of SAM was at the
highest levels in fractions 4, 5, and 6 (Fig. 2A), which
also contained high HGA-MT and XG-FT activity. We
also detected SAM uptake in other subcellular fractions
that contain endoplasmic reticulum markers and toward the bottom of the gradient at the mitochondrial
and plastid sedimentation points, suggesting that SAM
is also incorporated into these organelles in pea epicotyls. No detectable activity of mitochondrial or plastid
markers was detected in the Golgi fraction, indicating
that SAM uptake observed in the Golgi fraction was
not due to mitochondria or plastid present in that
fraction.
To further characterize the import of SAM into the
Golgi, we measured the uptake of two different radiolabeled forms of this substrate using enriched Golgi
vesicle fractions. One of these, [methyl-14C]SAM, is
radiolabeled on the methyl group that is transferred
onto HGA by HGA-MT, whereas the other, [carboxyl14
C]SAM, is radiolabeled on the carboxyl group that
remains in the S-adenosylhomocysteine (SAH) molecule following the methylation reaction. The results
shown in Figure 3 indicate that both substrates were
incorporated into Golgi vesicles but that the uptake of
[carboxyl-14C]SAM plateaued at 5 min, whereas the
uptake of [methyl-14C]SAM required 20 min to reach
this peak. Upon reaching a plateau, the incorporation
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Ibar and Orellana
Figure 2. Uptake of SAM and HGA-MT activity in
subcellular fractions of etiolated pea epicotyls. Pea
epicotyls were homogenized and the organelles separated in a discontinuous Suc gradient. Aliquots were
collected and utilized in the measurement of different
activities. A, SAM uptake measured in the presence
of 3 mM [methyl-14C]SAM for 5 min (d). Suc concentration was determined by refractometry ()). B,
HGA-MT activity measured in the presence of 4 mM
[methyl-14C]SAM and 6 mM cold SAM, 0.1% Triton
X-100, and 50 mg of polygalacturonic acid (HGA) in
STM (Suc-Tris-magnesium; n); cytochrome c oxidase
activity (mitochondrial marker; n). C, Glucosidase II
activity (endoplasmic reticulum marker; s); XG-FT
activity (Golgi marker; :). D, Immunoblot using antiTOC33 (plastid marker). The measurements were
done in duplicate and the average activity is plotted.
of [methyl-14C]SAM was found to be between 5- and
6-fold higher than [carboxyl-14C]SAM, suggesting that
when SAM is metabolized, the radiolabeled methyl and
carboxyl groups have a different fate within the Golgi
lumen.
To further analyze the differences observed during the
incorporation of the methyl- and carboxyl-radiolabeled
SAM substrates, we carried out a double-labeling experiment by incubating Golgi vesicles in the presence
of both [carboxyl-14C]SAM and [methyl-3H]SAM. As
shown in Table I, both radiolabeled substrates were incorporated into the Golgi vesicles but the methyl group
clearly accumulated at higher levels than the carboxyl
group, leading to a time-dependent increase in the 3H
to 14C ratio. These results suggest that SAM is transported into the lumen and that the methyl group is
then rapidly transferred onto endogenous acceptors. It
is therefore likely that the transfer of the methyl group
leads to the release of SAH, and our results further
suggest that SAH is not retained within the Golgi
lumen since the 3H to 14C ratio does not remain constant but increases with time.
The use of methyl-radiolabeled SAM in incorporation assays makes it difficult to distinguish between
the transport of SAM and the transfer of its donor
methyl group. To therefore characterize the transport
process alone, we performed experiments using only
[carboxyl-14C]SAM as the substrate to assess whether
SAM is truly transported into the Golgi lumen. We
evaluated the importance of the membrane integrity
during this importation process by treating the vesicles with 0.01% and 0.1% Triton X-100. This treatment
led to a strong decrease in the uptake of [carboxyl14
C]SAM (Fig. 4A), which was also found to be sensitive to temperature since it showed a 10-fold higher
activity at 25°C compared with 0°C. Boiling of the
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Import of S-Adenosylmethionine into the Golgi
formed after a 30-s incubation only. Our results show
that the uptake of SAM was saturable with an apparent Km of 33 mM (Fig. 5). We also measured the sensitivity of the uptake of [carboxyl-14C]SAM into Golgi
vesicles to osmotic changes as these will affect the vesicle volumes. The uptake of SAM was found to be lower
in the presence of a higher Suc concentration (Supplemental Table S1), a condition under which the volume
of the Golgi vesicles decreases. These results strongly
suggest that SAM is imported into the Golgi by an
active transporter.
The chemical structure of SAM resembles that of the
nucleotide sugars (NSs), and, since different NSTs have
been shown previously to be localized in the Golgi
apparatus in plants (Baldwin et al., 2001; Bakker et al.,
2005; Norambuena et al., 2005; Rollwitz et al., 2006), we
wanted to evaluate whether the transport of SAM is
also mediated by a NST-like transporter. We thus performed our SAM import assay in the presence of DIDS,
a general inhibitor of the NSTs (Wulff et al., 2000). Under conditions where DIDS produced a 10% to 15%
decrease in the uptake of SAM, the incorporation of
GDP-Fuc was found to be decreased by 70% (Fig. 6).
This result indicates that at the biochemical level, the
Golgi SAM transporter does not behave as a NST.
Because our data indicate that the methyl group of
SAM is transferred onto endogenous acceptors in the
lumen of the Golgi, we postulated that HGA was likely
to be one such acceptor. To test this possibility, we
incubated Golgi vesicles with [methyl-14C]SAM and
then adjusted the incubation media with TCA to a final
concentration of 10%, a treatment that sediments
both proteins and pectic polysaccharides (Goubet and
Mohnen, 1999b). The results showed that radioactive
material was indeed precipitated under these conditions (Fig. 7). Additionally, this radioactive material
was also precipitated by 70% ethanol, suggesting
that the methyl group is transferred onto polysaccharides (data not shown). To assess whether the TCAprecipitated radiolabeled material corresponded to
pectin, we separately treated this precipitate with pectinase and pectin methylesterase (PME) followed by a
reprecipitation with 10% TCA. As shown in Figure 7,
treatment with both enzymes decreased the amount of
radiolabeled material that could again be precipitated
with 10% TCA and at the same time increased the
amount of soluble radiolabeled material that was released into the supernatant. Since TCA also precipitates
proteins, we determined whether the TCA-precipitable
Figure 3. Time dependence of SAM uptake in Golgi vesicles. Golgi
vesicles (100 mg of protein) were incubated with 3 mM [methyl-14C]SAM
(A) or [carboxyl-14C]SAM (B) in a final volume of 100 mL for the times
indicated. The reaction was terminated by 10-fold dilution of the reaction and filtering the incubation medium through 0.45-mm cellulose
ester filters. The filters were washed with cold buffer, dried, and the
radioactivity associated with them estimated in a liquid scintillation
counter.
vesicles prior to the assay completely inactivated the
incorporation of SAM (Fig. 4A).
To obtain further evidence that a protein is involved
in this process, we treated the Golgi vesicles with trypsin and then measured the uptake of SAM. The results
of this experiment (Fig. 4B) showed that trypsinization
produces a decrease in the uptake of SAM (25%–30%).
The use of the same amounts of trypsin produced a
larger decrease (90%) in the HGA-MT activity levels
(Fig. 1C). These findings support the idea that a SAM
transporter is involved in the import of this substrate
into the Golgi and also indicates that the transport and
methyltransferase activities associated with SAM in
the Golgi are separate events.
To further characterize the transport of SAM, we
measured the substrate dependence of this process. In
addition, to minimize the contribution of the transfer
reaction to the measurement of radioactivity during
the uptake of [carboxyl-14C]SAM, the assay was per-
Table I. Ratio of 14C to 3H found in Golgi vesicles after incorporation of a mixture of
[carboxyl-14C]SAM 1 [methyl-3H]SAM
Vesicles (100 mg) were incubated with 3 mM SAM containing a mixture of [carboxyl-14C]SAM 1
[methyl-3H]SAM. After 30 s, 1 min, or 3 min of incubation, the reaction was stopped and filtered, and the
radioactivity associated with the filters was determined. The numbers in parentheses are pmoles of SAM
and are the average of triplicate measurements. The SD was less than 10%.
Medium
0s
30 s
1 min
3 min
1.00
(300/300)
1.14
(1.64/1.43)
1.71
(3.01/1.76)
2.22
(5.24/2.36)
2.45
(5.34/2.38)
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Ibar and Orellana
However, SAH also produced similar inhibitory effects upon HGA-MT activity (Supplemental Fig. S1),
and, since the transport of SAM and the transfer of the
methyl group are tightly associated, we cannot rule
out the possibility that the effects of this molecule on
the uptake of SAM are a consequence of its inhibition
of HGA-MT.
DISCUSSION
Figure 4. Effect of temperature, detergent, and proteases in the incorporation of SAM into Golgi vesicles. A, Golgi vesicles (100 mg of protein)
were incubated with 3 mM [carboxyl-14C]SAM for 5 min under different
conditions: 25°C; TX-100, incubation in the presence of 0.01% or 0.1%
(v/v) Triton X-100; 0°C, incubation in an ice bath; and 100°C, vesicles
boiled for 5 min prior to the assay. Incorporation was measured by
a filtration assay. B, Golgi vesicles (50 mg of protein) were treated for
10 min with 5 or 25 mg of trypsin, which was then blocked using a
specific inhibitor as described in ‘‘Materials and Methods.’’ The proteasetreated vesicles were then used to measure the uptake of SAM as described in Figure 4 by incubation with 3 mM [methyl-14C]SAM for 5 min.
material was sensitive to trypsin but found that treatment with this protease did not result in the release of
any radiolabeled material (Fig. 7). These results thus
suggest that SAM molecules that are incorporated into
Golgi vesicles are used as donor substrates for the transfer of methyl groups onto pectin but not protein acceptors.
Since many substrates involved in polysaccharide
biosynthesis are known to be transported into the Golgi
apparatus, we analyzed whether a combination of these
biosynthetic precursors had any effect in the uptake of
SAM. UDP-Glc had an inhibitory effect at high concentrations, whereas ADP-Glc and L-Met showed only
minor inhibitory effects. In contrast, ATP, acetyl-CoA,
UDP-GalA, and UDP-GlcA showed no effects upon
the uptake of SAM. Interestingly, different combinations
of substrates likely to be involved in HGA biosynthesis, such as UDP-GalA 1 acetyl-CoA or UDP-GlcA 1
acetyl-CoA, appeared to cause slight increases in the
uptake of SAM (Fig. 8). Significantly, SAH, the principal by-product of the HGA-MT/SAM methyltransferase reaction, showed the strongest inhibitory effects
upon the uptake of SAM into Golgi vesicles. This inhibitory effect of SAH has also been observed in the
transport of SAM into chloroplasts and mitochondria.
The methylation of HGA occurs in the Golgi apparatus, and biochemical analyses have indicated that
the catalytic domains of the methyltransferases involved in this process face the lumen of this organelle
(Goubet and Mohnen, 1999a; this study). Proteomic
analyses of cellular organelles have identified a number of putative Golgi methyltransferases (GMTs), two
of which (GMT1 and GMT2) have now been confirmed
to be localized in the Golgi apparatus (Dunkley et al.,
2006). Hydropathy analysis of these proteins has further indicated that they are type II membrane-bound
proteins, supporting the idea that most of the enzyme,
including its active site, faces the lumen of the Golgi.
Although none of the candidate GMTs has yet been
functionally tested, it is likely that some of them are
involved in the methylation of HGA in the lumen of
the Golgi. Recently, Mouille et al. (2007) showed that
the QUA2 locus from Arabidopsis (Arabidopsis thaliana) encodes a putative methyltransferase that is Golgi
localized. A QUA2 mutant exhibited a decrease in
HGA, and the corresponding gene is coexpressed with
GAUT1, a galacturonosyltransferase involved in HGA
biosynthesis (Sterling et al., 2006). In addition, the
QUA2 gene encodes a putative type II membrane protein that contains a methyltransferase domain that
would necessarily face the lumen of the Golgi. These
findings strongly indicate that the methyltransferases
involved in HGA biosynthesis harbor a catalytic domain that faces the lumen of the Golgi apparatus and
Figure 5. Initial rates of SAM uptake as a function of external [carboxyl14
C]SAM concentrations in Golgi vesicles. Golgi vesicles (100 mg) were
incubated with various concentrations of [carboxyl-14C]SAM and incorporation into Golgi vesicles was estimated by a filtration assay. The
curve was fitted with the Michaelis-Menten equation, thus giving Km
and Vmax values of 33 mM and 227 pmol/mg 30 s.
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Import of S-Adenosylmethionine into the Golgi
that the methylation of these polysaccharides takes place
in this compartment.
SAM is a methyltransferase substrate and it is widely
accepted that the enzymes involved in its biosynthesis
are exclusively located in the cytosol (Schröder et al.,
1997; Ravanel et al., 1998; Hanson and Roje, 2001).
Hence, for the methylation of HGA, SAM must be imported into the lumen of the Golgi. In this study, we
provide different lines of evidence indicating that SAM
is indeed transported into the lumen of the Golgi. These
include data showing the following: (1) by subcellular
fractionation analysis, strong levels of SAM uptake
occur in the Golgi fraction; (2) the uptake of SAM is
dependent upon the membrane integrity of the Golgi
vesicles as treatments with detergent inhibit this process; (3) SAM uptake into the Golgi is saturable with
an apparent Km of 33 mM; (4) SAM uptake is sensitive to
both temperature and osmotic changes that alter the
volume of the Golgi vesicles; and (5) trypsin treatments inhibit SAM uptake. Taken together, these data
strongly support our conclusion that SAM is transported into the Golgi lumen and that a SAM transporter is localized at the membrane of this organelle.
SAM transport activity has been detected previously
in mitochondria and plastids (Horne, et al., 1997;
Ravanel et al., 2004; Bouvier et al., 2006). The Km for
the SAM transporter from plastids is 38 mM, which is
very similar to the Km value we obtained in our present
analysis for the uptake of SAM by the Golgi vesicles
(33 mM). We do not yet know what the free cytosolic
concentration of SAM is, and it is thus difficult to predict whether its transporter is working under saturating
conditions. This is an important issue to be elucidated
in the future because, depending on the concentration
of SAM in the cytosol, this transporter may be a limiting step in the methylation process.
The uptake of SAM was found to be inhibited by
SAH, which had also been found to be the case for
other SAM transporters. However, we found that the
activity of HGA-MT was also inhibited by SAH, and
therefore cannot yet exclude the possibility that the
inhibitory effects of this molecule are not due to a direct effect upon the transporter but are in fact caused
Figure 6. SAM uptake has low sensitivity to DIDS. Golgi vesicles (100
mg) were incubated with 3 mM [methyl-14C]SAM or 1 mM GDP-[3H]Fuc
by 5 min at 25°C in the presence or absence of 50 mM DIDS. The SAM
uptake was measured using a filtration assay.
Figure 7. Methylated products obtained upon incubation with SAM are
sensitive to enzymes that degrade pectins. Golgi vesicles (100 mg of
protein) were incubated with 3 mM [methyl-14C]SAM for 60 min. Immediately after, polysaccharides and proteins were precipitated by
adding 1 volume of 20% TCA. The precipitated fraction was in buffers
with different pH (described in ‘‘Materials and Methods’’) and treated
with pectinase (pH 4.0; A), PME (pH 7.5; B), or trypsin (pH 7.5; C) for 30
min and 4 h. Then, the polysaccharides and proteins were reprecipitated
with a volume of 20% TCA, and the radioactivity remaining both in the
supernatant and pellet was determined by scintillation counting. Experiments were done in triplicate.
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Ibar and Orellana
Figure 8. Effect of different substrates in the uptake of SAM by Golgi
vesicles. Golgi vesicles (100 mg of protein) were incubated for 10 min
with increasing concentrations (0, 5, 10, 50, 100 mM) of different compounds. Then, the vesicles were incubated with 3 mM [methyl-14C]SAM
for 5 min in urea final volume of 100 mL. The incorporation of SAM was
measured using a filtration assay. The values are expressed as a percentage of the SAM uptake activity determined in the absence of any of
the compounds used in the experiment. The highest value on each case
corresponds to (mean 6 SE): A, 35.92 6 0.97 pmol mg21 5 min21; B,
30.23 6 0.19 pmol mg21 5 min21; C, 42.73 6 2.10 pmol mg21
5 min21; D, 42.73 6 2.10 pmol mg21 5 min21; E, 41.38 6 1.78
pmol mg21 5 min21; F, 32.89 6 2.61 pmol mg21 5 min21; G, 44.11 6
2.02 pmol mg21 5 min21; H, 34.73 6 1.66 pmol mg21 5 min21; I, 38.44 6
4.44 pmol mg21 5 min21; and J, 36.99 6 6.93 pmol mg21 5 min21.
incorporation of SAM, but the addition of acetyl-CoA
in conjunction with either UDP-GalA or UDP-GlcA marginally stimulated SAM uptake. This result suggests
that a coordinated uptake of the substrates involved in
the biosynthesis of pectins may occur in the Golgi
apparatus.
With regards to the putative existence of a SAM transporter, another family of Golgi-localized transporters
has already been described. These are the NSTs that are
responsible for the import of glycosyltransferase substrates, and the catalytic domains for these enzymes
also face the Golgi lumen. It is noteworthy that the
chemical structure of SAM shows some similarity with
NSs, particularly at the nitrogenated base that is present in both structures. Hence, one of the questions that
arises is whether SAM may in fact be transported by a
protein related to NSTs. However, we tested the effect
of DIDS, an inhibitor of anionic transporters that has
been shown to inhibit NSTs (Wulff et al., 2000), and
found that it showed little effect upon the uptake of
SAM in Golgi vesicles.
The genes encoding SAM transporters have now been
cloned in yeast, human, and Arabidopsis (Marobbio
et al., 2003; Agrimi et al., 2004; Bouvier et al., 2006;
Palmieri et al., 2006), and belong to the mitochondrial
carrier family. One of the interesting features of these
carriers is that they are inhibited by SAH. We also found
that SAH was a strong inhibitor of SAM uptake into
Golgi vesicles. In addition, these characterized transporters work as antiporters; therefore, it is likely that
the Golgi-localized transporter may also work as an
antiporter, taking the SAH produced in the lumen of the
organelle back to the cytosol. Hence, it is likely that a
similarity may exist between the putative SAM Golgilocalized transporter and those located in mitochondria
and plastids. Significantly in this regard, phylogenetic
analyses of the SAM transporters and the NSTs show
that they form different clusters, suggesting that the
putative Golgi-localized SAM transporter may be encoded by a member of the mitochondrial carrier family.
Future studies should lead us to the identification of
this gene.
MATERIALS AND METHODS
by a failure in the transfer of methyl groups that ultimately leads to a decrease in the transport of SAM. UDPGlc was also found to have inhibitory effects upon SAM
uptake at high concentrations, but none of the other
NSs that we tested has this effect. We do not yet know
the reason for the inhibition of this process by UDPGlc, but it does not seem to be a general effect of UDPsugars since none of the other NSs inhibited the import
of SAM into the Golgi. Whether this effect is due to
some negative interaction between glucosylation reactions in the Golgi and the incorporation of SAM
remains to be determined.
Neither UDP-GalUA nor UDP-GlcUA, both substrates of the pectin biosynthesis pathway, affected the
Chemicals
The chloride salt of SAM, the ammonium salt of UDP-GalA, SAH, acetylCoA, ATP, L-Met, Triton X-100, trypsin and trypsin inhibitor, polygalacturonic
acid (referred to as HGA in this article), pectinase from Rhizobium sp. (crude
powder), and PME from orange peel were each purchased from Sigma.
[Methyl-14C]SAM (specific activity 52.7 mCi/mmol) and GDP-[3H]Fuc (specific
activity 20.0 Ci/mmol) were purchased from New England Nuclear. [Carboxyl14
C]SAM (specific activity 62 mCi/mmol) and [methyl-3H]SAM (specific activity 84.0 Ci/mmol) were obtained from Amersham Pharmacia. Cellulose
ester filters (0.45 mm) were purchased from Millipore.
Plant Material
Pea (Pisum sativum var. Alaska) seeds were obtained from ANASAC and
grown in moist vermiculite for 7 or 8 d in the dark at 25°C. Stem segments (1 cm)
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Import of S-Adenosylmethionine into the Golgi
were excised from the elongating region of the epicotyls and kept on ice until
homogenization.
Subcellular Fractionation and Enrichment for
Golgi Vesicles
Vesicles were obtained essentially as described previously by Muñoz et al.
(1996). Briefly, pea stems were homogenized using razor blades in the presence of 1 volume of 0.5 M Suc, 0.1 M KH2PO4, pH 6.65, 5 mM MgCl2. The homogenate was then filtered through Miracloth (Calbiochem) and centrifuged
at 1,000g for 5 min. The supernatant was loaded onto a 1.3 M Suc cushion and
centrifuged at 100,000g for 90 min. The upper phase was then removed without disturbing the interface fraction. A discontinuous gradient was then formed
on top of the interface fraction by the addition of 1.1 and 0.25 M Suc cushions,
followed by centrifugation for 100 min at 100,000g. The interface 0.25/1.1 M
Suc fraction was then collected, diluted in 1 volume of distilled water, and centrifuged at 100,000g for 50 min. The pellet was resuspended using a Dounce
homogenizer in buffer containing 0.25 M Suc, 1 mM MgCl2, and 10 mM TrisHCl, pH 7.5 (STM buffer). The vesicles were kept at 280°C until use. All of
these procedures were performed on ice or at 4°C. The UDPase latency of the
vesicles varied between 75% and 90% for different preparations.
Enzymatic Assays and Organelle Markers
XG-FT activity was measured at 25°C for 30 min in a final volume of 100 mL,
as described by Wulff et al. (2000). Briefly, the reaction contained 1 mM GDP[3H]Fuc, 100 mg of tamarind XG, 10 mM HEPES/KOH, pH 7.0, 10 mM MnCl2,
and 0.05% (v/v) Triton X-100 and was terminated after 30 min by adding 250
mL of 95% (v/v) ethanol. The samples were kept on ice for 30 min, and then
filtered through 0.45-mm cellulose ester filters using a filtration system (model
FH225V; Hoefer). The filters were then washed three times with 1 mL of 70%
(v/v) ethanol containing 1 mM EDTA and dried. The radioactivity was estimated by liquid scintillation counting. To estimate the XG-FT activity in permeabilized vesicles, we added 0.05% (v/v) Triton X-100 to the incubation medium.
The HGA-MT assay was modified from the method described by Goubet
et al. (1998). Briefly, Golgi vesicles (50 mg of protein) or gradient aliquots were
incubated in a final volume of 50 mL containing 6 mM [methyl-14C]SAM (final
concentration), 0.25 M Suc, 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2 (STM buffer)
at 30°C for 1 h. The reaction was then stopped and the methylated products
precipitated by the addition of one volume of 20% (w/v) TCA and 5 mL of a
10% (w/v) bovine serum albumin solution. The resulting suspension was
centrifuged for 10 min at 4,000g. Unincorporated SAM was removed by
washing the pellets twice with 200 mL of 2% (w/v) TCA. The washed pellets
were boiled for 10 min with 25 mL of 2 M NaOH, neutralized with 30 mL of 2 M
HCl, and the radioactivity incorporation measured by liquid scintillation.
To measure glucosidase II activity, aliquots of 1 mL from the gradients were
centrifuged with 1 volume of water for 60 min at 100,000g. The pellets obtained
were then resuspended in 100 mL of STM buffer. Glucosidase II activity was
determined as described by Trombetta et al. (1996) using 50 mL of resuspended
pellet with 1 volume of distilled water in the presence of 20 mM HEPES, pH 7.5,
5 mM p-nitrophenyl-a-glucopiranoside, and Triton X-100 0.02% for 30 min at
room temperature. The reaction was terminated by adding 300 mL of 2 M Tris
base containing 2% SDS and the absorbance was then measured at 405 nm.
To determine cytochrome c oxidase, 40 mL of the gradient fractions were
used to measure the activity as described by Briskin et al. (1987).
TOC 33 was determined by western blot using a specific antibody kindly
donated by Dr. Danny Schnell.
Proteolysis of Golgi Vesicles
Golgi vesicles (200 mg of protein) were incubated with 5 or 25 mg of trypsin
in a solution containing 0.25 M Suc, 1 mM MgCl2, and 10 mM Tris-HCl, pH 7.5,
for 10 min at 30°C in a final volume of 25 mL. These reactions were also
incubated in the presence or absence of 0.02% (v/v) Triton X-100 and were
stopped by the addition of 1.25 mL of a 10 mg/mL concentration of soybean
trypsin inhibitor, followed by an incubation for 10 min at 30°C. As a control,
only the trypsin inhibitor was added. The samples were kept in ice until the
enzymatic reactions were performed.
Uptake of SAM and GDP-Fuc into Golgi Vesicles
Golgi vesicle preparations (100 mg of protein) were incubated for 5 min at
25°C (or as indicated) with 3 mM [methyl-14C]SAM or [carboxyl-14C]SAM in
100 mL of a buffer containing 0.25 M Suc, 1 mM MgCl2, and 10 mM Tris-HCl, pH
7.5. The incubation was terminated by dilution with 10 volumes of an ice-cold
solution containing 0.25 M Suc, 1 mM MgCl2, and 10 mM Tris-HCl, pH 7.5, and
immediately filtered through 0.45-mm cellulose ester filters using the filtration
system described above. The filters were then washed with an additional
10 volumes of the same solution, dried, and subjected to liquid scintillation
counting.
The uptake of GDP-Fuc was performed using a filtration assay as described previously by Wulff et al. (2000). Golgi vesicles (100 mg of protein)
were incubated with 1 mM GDP-[3H]Fuc in the same incubation buffer used for
the SAM uptake assay. The reaction was terminated in the same manner as
described above and the radioactivity associated with the filters was determined by liquid scintillation counting.
Enzymatic Treatments
Golgi vesicles (50 mg of protein) or aliquots from the gradients were
incubated at 30°C for 1 h in 50 mL of a solution containing 6 mM [methyl14
C]SAM, 0.25 M Suc, 10 mM Tris-HCl, pH 7.5, and 1 mM MgCl2. The reaction
was stopped and the methylated products precipitated by the addition of
1 volume of 20% (w/v) TCA and 5 mL of a 10% (w/v) bovine serum albumin
solution. The resulting suspension was centrifuged for 10 min at 4,000g. The
TCA insoluble fraction was resuspended in 50 mL of 0.1 M NH4Ac, pH 4.0, for
pectinase treatment or 0.1 M Tris-Cl, pH 7.5, for PME or trypsin treatment. For
these analyses, either 0.3 units of pectinase, 1 unit of PME, or 25 mg of trypsin
was added to the samples, followed by an incubation for both 30 min and 4 h
at 25°C for pectinase or 30°C for PME and trypsin. The reaction was stopped
by adding 1 volume of 20% TCA. The samples were then centrifuged for
15 min at 10,000g, and the radioactivity associated with the pellet and the
supernatant was determined by liquid scintillation counting.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Effect of SAH in the incorporation of SAM into
Golgi vesicles and HGA-MT activity.
Supplemental Table S1. Incorporation of SAM is sensitive to osmotic
changes.
ACKNOWLEDGMENT
We are very grateful to Francisca Reyes for her help with the subcellular
fractionation experiments.
Received June 26, 2007; accepted August 23, 2007; published August 31, 2007.
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