ATP-binding cassette-like transporters are involved in
the transport of lignin precursors across plasma and
Yu-Chen Miao and Chang-Jun Liu1
Biology Department, Brookhaven National Laboratory, Upton, NY 11973
Edited by Ronald R. Sederoff, North Carolina State University, Raleigh, NC, and approved November 11, 2010 (received for review June 1, 2010)
Lignin is a complex biopolymer derived primarily from the conden-
sation of three monomeric precursors, the monolignols. The syn-
thesis of monolignols occurs in the cytoplasm. To reach the cell wall
where they are oxidized and polymerized, they must be trans-
ported across the cell membrane. However, the molecular mecha-
nisms underlying the transport process are unclear. There are
conflicting views about whether the transport of these precursors
we know little about what chemical forms are required. Using
isolated plasma and vacuolar membrane vesicles prepared from
Arabidopsis, together with applying different transporter inhibi-
derivatives by these native membrane vesicles. We demonstrate
that the transport of lignin precursors across plasmalemma and
port processes, involving ATP-binding cassette-like transporters.
Moreover, we show that both plasma and vacuolar membrane
vesicles selectively transport different forms of lignin precursors.
In the presence of ATP, the inverted plasma membrane vesicles
preferentially take up monolignol aglycones, whereas the vacuolar
vesicles are more specific for glucoconjugates, suggesting that the
chemical forms in conveying them to distinct sites, and that gluco-
sylation of monolignols is necessary for their vacuolar storage but
not required for direct transport into the cell wall in Arabidopsis.
derived from the condensation of three monomeric pre-
cursors, p-hydroxyphenyl, coniferyl, and sinapyl alcohols (termed
monolignols). Although lignin affords vital structural support to
terrestrial plants and provides hydrophobicity to their vascular
elements, its presence in cell walls constitutes a formidable ob-
stacle for digesting forage crops, pulping, and producing re-
newable biofuels from cellulose and hemicelluloses (1, 2).
Monolignols are synthesized in the cytosol. Thereafter, these
monomeric precursors are exported into the cell wall, where
they are polymerized and integrated into the wall to form p-
hydroxyphenyl, guaiacyl, and syringyl subunits (3). Accordingly,
monolignol transport across plasma membranes is a critical step
affecting the deposition of lignin and the thickening of the sec-
precursors, the molecular mechanisms underlying their sub-
cellularsequestration andextracellulartransportation aresketchy
(3). Earlier investigations in gymnosperms and angiosperms pro-
vided conflicting interpretations. Using [3H]Phe to label the de-
veloping xylem, several studies found that the radiolabel was
associated with the rough endoplasmic reticulum (ER) and the
Golgi body, and also with some vesicles fused with the plasma
membrane (4–6). The potential vesicular trafficking between the
cytosol and plasmalemma in differentiating tracheids of xylem
tissues was also reported (4). These autoradiographic and ultra-
structural analyses engendered the assumption that the lignin
exocytosis. However, Kaneda et al. (7) recently adopted a new
approach to preparing labeled xylem cells of lodgepole pine for
autoradiographic studies. Using cryofixation and freezing sub-
ignin is a complex and irregular biopolymer that is primarily
stitution techniques, they substantially minimized the damage in
sectioned cells, thus preventing the misinterpretation of autora-
diography. Then, feeding dissected xylem tissue with a [3H]Phe
radiotracer and selectively inhibiting phenylpropanoid and pro-
tein biosynthesis by different inhibitors, they discovered that the
radiolabel in the ER-Golgi was primarily incorporated into pro-
and Golgi-vesicle clusters abundant in the developing xylem cells
ER-Golgi-vesicle-mediated exocytosis does not play a major role
in transport of the monolignols (7).
Genetic and chemical analyses demonstrated that lignin bio-
synthesis displays considerable plasticity. Besides the three clas-
sical monolignols, some nontraditional phenolic monomers are
incorporated into lignin under certain circumstances (8, 9). For
example, in a natural cinnamyl-alcohol dehydrogenase (CAD)-
deficient mutant of pine and transgenic tobacco knocked down in
CAD, hydroxycinnamaldehydes were incorporated into lignin
(10). Similarly, a lack of the caffeic acid O-methyltransferase in
a maize bm3 mutant caused the accumulation of 5-hydrox-
yconiferyl alcohol and the buildup of this unusual precursor in
lignin (11). In addition, lignins are frequently acylated with ace-
tate or p-coumarate (12, 13); such acylation implicates the in-
corporation of acylated lignin monomers. The accommodation
of alternative monomers in lignification led to the suggestion of
nonspecific passive diffusion of lignin precursors across the
plasma membrane (14). This notion was supported by the ob-
servation of the in vitro partitioning of lignin monomers or ana-
logs by immobilized liposomes and/or lipid-bilayer discs (15, 16).
Although lignin biosynthesis displays considerable flexibility in
incorporating different monomeric precursors, many studies
note that these monomers are deposited differentially in discrete
regions of particular tissues or cells. For example, lignin in the
cell walls of vessels in birch wood is derived mainly from con-
iferyl alcohol, whereas its fiber wall incorporates both sinapyl
and coniferyl alcohols (17). Similarly, in Arabidopsis stems, the
lignin of the vascular bundle in vessels primarily contains
guaiacyl lignin (from coniferyl alcohol), whereas the inter-
fascicular fibers are enriched in syringyl units (from sinapyl al-
cohol) (18). Moreover, when feeding the labeled monolignols
into the developing xylem, the radiolabeled p-coumaryl alcohol is
preferentially laid down in the middle lamella/cell corners,
whereas coniferyl alcohol is mainly located within the secondary
wall (19). These data suggest that the biosynthesis and deposition
of lignin monomers into cell wall is a highly organized, regulated
process, and that active transportation mechanisms might se-
lectively permit the deposition of the particular monolignols.
Author contributions: C.-J.L. designed research; Y.-C.M. performed research; Y.-C.M. and
C.-J.L. analyzed data; and Y.-C.M. and C.-J.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 28, 2010
| vol. 107
| no. 52 www.pnas.org/cgi/doi/10.1073/pnas.1007747108
Besides depositing lignin monomers into the cell wall, gym-
nosperms and some angiosperms store a significant fraction of
monolignol 4-O-glucosides within the cytoplasm, presumably in
the cell’s vacuoles (3). The ability of those plants to divert
monolignols to a storage compartment rather than directly in-
corporating them into cell-wall polymers also implies that the
plant contains proteins that can transport monolignols or their
derivatives across lipid bilayers into particular compartments.
Recently, several families of plant membrane transporters,
including ATP-binding cassette (ABC) transporters and multi-
drug and toxic compound extrusion (MATE) transporters, were
shown to be involved in sequestering intracellularly a variety of
small molecular compounds, including phenolics (20, 21). Global
transcriptomic and proteomic studies in gymnosperms and
angiosperms frequently reveal the presence and high expression
of some membrane transporters in lignified wood tissues (22, 23).
All such studies suggest that these membrane transporters may
be active in sequestration and transport of the monolignols.
However, direct biochemical evidence has been lacking.
In this study, we isolated plasma and vacuolar membrane
vesicles from Arabidopsis young rosette leaves and the roots of
poplar (Populus tremuloides). With the prepared membrane
vesicles, we undertook in vitro uptake assays for monolignols,
their glucosides, and related phenolics. Together with the ap-
plication of different transporter inhibitors, we reveal that the
transport of lignin monomeric precursors across both plasma
and vacuolar membranes is an active ATP-dependent process.
Omitting ATP or including specific ABC-type transporter inhibi-
tors severely impaired the transport activity of plasma or vacu-
olar membrane vesicles to monolignols or their glucosides. In the
presence of ATP, plasma membrane vesicles selectively transport
monolignol aglycones, whereas vacuolar vesicles prefer mono-
lignol 4-O-glucosides, implying that different ABC-like trans-
porters recognize and convey distinct chemical forms of lignin
precursors to particular sites.
ATP-Dependent Transport of Monolignols by Arabidopsis Plasma
Membrane Vesicles. To mimic the in vivo efflux of lignin pre-
cursors across plasmalemma, we prepared inside-out (inverted)
plasma membrane vesicles from Arabidopsis rosette leaves. We
first used an aqueous polymer two-phase partitioning procedure
(24) to isolate right-side-out plasma vesicles from an Arabidopsis
microsomal fraction. Subsequently, we treated the vesicles with
the detergent Brij 58 (25) to convert the right-side-out vesicles to
the inside-out ones (cytoplasmic-side-out). We monitored the
quality of our membrane preparation by Western blots using
antibodies against plasma membrane H+-ATPase, vacuolar H+-
pyrophosphatase (V-PPase), and ER luminal-binding protein
(Bip) of Arabidopsis thaliana. The gel blot showed that the
plasma membrane vesicles were predominantly enriched in the
upper phase of the two-phase partition (Fig. 1A). The inverted
vesicles showed high H+-ATPase activity, exceeding by approx-
imately three- to fivefold the latent activities of the crude
microsomes and the right-side-out vesicles; this activity was
inhibited severely by sodium ortho-vanadate, a suppressor of
ATPase activity (26) (Fig. 1B). These data verify the high quality
of our prepared inverted plasma membrane vesicles.
The inverted vesicles were incubated with monolignols, repre-
We then collected the vesicles by vacuum filtration through a wet
cellulose-nitrate membrane filter. After thoroughly rinsing the
filters,we re-extractedthecompounds retainedwithin thevesicles
and examined them by HPLC. The amount of monolignols taken
up by and recovered from the vesicles clearly depended upon the
ATP molecules added during incubation. In the absence of
MgATP, only low amounts of monolignols were detected (Fig.
1C; Fig. S1). Adding MgATP in the assay medium increased the
uptake of coniferyl alcohol more than threefold (Fig. 1C). We
observed a similar ATP-dependent uptake when the inverted
plasma membrane vesicles from Populus were used (Fig. S2A).
We explored further the uptake of coniferyl alcohol by the
inverted plasma vesicles in a time-course experiment. The trans-
port of monolignols in the presence of MgATP rose with in-
cubation time, and gradually reached its maximum after 1 h (Fig.
1D). This behavior likely coincides with the gradual depletion of
To determine whether the transport of monolignols across
plasma membranes specifically depends on ATP, we replaced
ATP with other nucleotides such as MgGTP, -TTP, and -CTP.
ATP was the most effective nucleotide triphosphate in driving
monolignol transport, whereas GTP, TTP, and CTP slightly
promoted the transport of coniferyl alcohol (Table S1).
coniferyl alcohol across plasmalemma is primarily an energy-
dependent process, although some non-energy-dependent trans-
port was evident in the in vitro assay.
Kinetics of Monolignol Uptake by Plasma Membrane Vesicles. The
uptake of coniferyl alcohol by the inverted plasma membrane
vesicles exhibited typical Michaelis–Menten-type kinetics. At
a fixed concentration of MgATP, we calculated the apparent Km
and Vmaxvalues for transport of coniferyl alcohol as, respectively,
71.4 μM and 344 pmol·mg protein−1·min−1(Fig. 1E), and the
calculated Kmvalue for ATP was 468 μM (Fig. 1F). These num-
bers are close to those observed for other ABC-type transporters
involved in the transport of low-molecular-weight organic com-
pounds (27, 28).
Effects of Transport Inhibitors on the Uptake of Monolignols Across
the Plasma Membrane. Theinvertedplasmamembranevesicleswere
pretreated with the following inhibitors. Sodium ortho-vanadate
is a typical suppressor of ATPase activity and inhibitor of ABC
transporters that acts as a phosphate analog (26). Verapamil and
nifedipine are general Ca2+-channel blockers, also known to pref-
erentially inhibit ABCB-type ABC transporters (27, 29). Glyben-
especially ABCC-type ABC transporters (27, 30). All impaired the
and nifedipine reduced transport activity by ≈60%, reaching the
baseline level of uptake in the absence of ATP (Fig. 2). In contrast,
ionophores/protonophores, such as gramicidin D which dissipates
both the pH gradient and membrane potential, and nigericin and
NH4Cl which destroy the pH gradient across membranes (31), dis-
played little effect on monolignol uptake (Fig. 2).
Selective Transport of Phenolics by Plasma Membrane Vesicles. We
testedvariousphenolics aspotential substrates inuptake assaysof
plasma membrane vesicles. These include hydroxycinnamic acids,
hydroxycinnamyl aldehydes, alcohols, and/or their glucosides
(Table 1). The inverted vesicles showed a base level and unse-
lective transport activity to a range of phenolic aglycones in the
absence of ATP, indicating potential intrinsic, nonselective per-
meability of the plasmalemma to those hydrophobic compounds.
When we added ATP, the transport activity toward hydrox-
ycinnamyl alcohols and aldehydes profoundly increased. However,
the inverted membrane vesicles did not display any measurable
transport activity for the monolignol glucosides coniferin and
syringin, and only showed a negligible uptake of ferulic acid, either
in the absence or presence of MgATP (Table 1).
Uptake of Monolignol Glucosides into Arabidopsis Vacuolar Vesicles.
Arabidopsis accumulates soluble monolignol 4-O-glucosides in its
root tissues, presumably in the vacuoles of cells (32). We asked
whether the vacuolar sequestration and accumulation of mono-
lignol glucosides involve active transport.
We prepared vacuolar membrane vesicles from intracellular
membranes, derived from two-phase partitioning, by sucrose
differential-density centrifugation. The quality of membrane
separation was monitored by examining marker proteins with
immunoblots against V-PPase, H+-ATPase, and Bip antibodies
for the selected centrifugation fractions (Fig. 3A), and by mea-
Miao and Liu PNAS
| December 28, 2010
| vol. 107
| no. 52
suring the activities of V-PPase for PPi hydrolysis and PPi-
dependent H+translocation and of H+-ATPase (Fig. S3). We
collected the fractions enriched with vacuolar membranes that
showed high activity and strong immune signals for V-PPase, and
used them for the uptake assays.
Monolignol 4-O-glucosides were prepared by enzymatic reac-
UGT72E2, one of the functionally characterized glycosyltrans-
ferases from Arabidopsis (33) (Fig. S4).
We found that when monolignol glucosides were incubated
the presence of MgATP (Fig. 3B). The ABC transporter inhibitor
vanadate impaired this transport activity severely, but gramicidin
D, a compound potentially inhibiting MATE-transporter activity
(31), had little effect (Fig. 3C).
In contrast to the relaxed specificity of plasma membranes for
phenolics, the uptake of vacuolar vesicles showed strict substrate
occurred for the 4-O-glucosides coniferin and syringin. The vacu-
olar membranes exhibited only minor transport activity for mono-
lignol aglycones, whether ATP was present or absent; no activity
was detected toward other phenolics (Table 1).
Similar to the kinetic behavior of plasma membranes trans-
porting monolignol aglycones, the vacuolar vesicles sequestering
monolignol glucosides also displayed Michaelis–Menten-type
kinetics. The apparent Kmfor coniferin was 113.9 μM, and was
1 mM for ATP (Fig. 3 D and E).
ABC-like Transporters Export Lignin Precursors Across the Plasma-
lemma and Sequester Them into Vacuoles. The molecular mecha-
nisms for depositing monolignols or their derivatives from the
cytoplasm into the cell wall and for sequestering them intracel-
lularly have been long-standing issues. Because of its amphiphilic
property, the lipid-bilayer membrane may be permeable to hydro-
phobic phenolic compounds. An in vitro partitioning assay readily
detected lignin monomeric analogs absorbed into immobilized
liposomes or lipid-bilayer discs (15, 16), supporting a nonselective,
passive diffusion mechanism for monolignols (14).
However, using plasma and vacuolar membranes from Arabi-
dopsis and Populus, which retain many physiological properties of
the native lipid bilayers, to mimic the efflux of lignin precursors
across plasmalemma and their influx into vacuoles, we demon-
strated that significant movement of monolignols or their gluco-
sides into the inverted plasma or vacuolar vesicles, respectively,
1 / V
1 / [S]
1 / [S]
1 / V
30 40 50 60
crude right-out PM
Absorbance at 280 nm (mAU)C
(nmol / mg protein)
Coniferyl alcohol ( M)
(pmol / mg protein / min)
(pmol / mg protein / min)
of monolignols by inside-out plasma membrane
fractions by probing with antibodies against Ara-
bidopsis plasma membrane H+-ATPase (PM-H+-
ATPase), vacuolar H+-pyrophosphatase (V-PPase,
tonoplast marker), and endoplasmic reticulum-
binding protein (Bip, ER marker). (B) H+-ATPase
activities of different membrane preparations in
from three replicates. (C) Portions of HPLC traces
showing the recoveredconiferylalcohol fromthe
inverted vesicles in the uptake assay in the pres-
ence (a) and absence (b) of ATP, the presence of
sodium vanadate and ATP (c), and the absence of
phenolic substrate (d). (D) Time course of the
transport of coniferyl alcohol into the inside-out
vesicles. The results are the mean and SD of
two replicates. (E and F) The kinetics of transport
of coniferyl alcohol into Arabidopsis inside-out
plasma membrane vesicles for coniferyl alcohol
(E) and MgATP (F) concentrations. The data are
plotted by a nonlinear regression analysis fit
to the Michaelis–Menten equation. The insets in
E and F show Lineweaver–Burk plots. The data
are the means and SD of two or three replicates.
Preparation and characterization of
| www.pnas.org/cgi/doi/10.1073/pnas.1007747108Miao and Liu
occurred under energized conditions (Figs. 1 and 3; Table 1). In
the absence of ATP, lignin precursors were transported by plasma
or vacuolar membrane vesicles only at a low level (Figs. 1 and 3;
diffusion that may function as a component in transporting lignin
monomeric precursors across membranes. However, passive dif-
fusion is unlikely to play a major role; instead, transport across
plasma and vacuolar membranes is dominated by ATP-dependent
primary transport. Several lines of evidence corroborated our
conclusion. First, the uptake of monolignols or their glucosides
into vesicles depended upon nucleotide phosphates, particularly
ATP (Table S1). Second, uptake showed selectivity for different
substrates. In the presence of ATP, plasma vesicles preferentially
transport monolignol aglycones, whereas vacuolar sequestration
or their glucosides displays typical Michaelis–Menten kinetics
(Figs. 1 E and F and 3 D and E), or a cooperative ligand-binding
behavior observed in the uptake of Populus membrane vesicles
(Fig. S2B), indicating a membrane-protein-mediated biochemical
process rather than passive diffusion. Last, a set of ABC trans-
precursors transported across the plasmalemma and sequestered
into vacuoles require membrane-protein-mediated active trans-
port that involves ATP-binding cassette-like transporters.
uptake of monolignols by the plasma membrane vesicles in the
of the respiratory electron-transport chain that depletes ATP (34),
it also can directly damage cell membranes by altering the mem-
brane’s resistance (increasing its permeability) (35). We tested this
possibility by monitoring the potential changes of the latent ATP-
after KCN treatment. In the presence of ATP, we observed that
of the right-side-out membrane vesicles but did not affect the ac-
tivity presented in the inside-out membranes (Fig. S5), indicating
permeability of the membranes (for ATP) or damage the integrity
of the membrane vesicles. Although the underlying mechanism of
the inhibition by KCN on monolignol uptake may be complex, one
possibility for the observed effect might reflect the action of KCN
retention of transported phenolics in the vesicles.
Compared with vacuolar vesicles, plasma membranes dis-
played notable promiscuity in conveying different phenolics in
the presence or absence of ATP molecules (Table 1). The lesser
selectivity of plasma membranes might explain the observed
plasticity of lignin biosynthesis. The promiscuous active transport
and/or the low level of intrinsic diffusion may lead to the de-
position of nonclassic lignin precursors into the cell wall.
Different Structural Forms of Monolignols Are Required for Transport
Across Plasmalemma and Sequestration into Vacuoles. In gymno-
sperms and some angiosperm species, monolignols often are glu-
cosylated on the phenolic hydroxyl group to form 4-O-β-D-
glucosides, namely coniferin and syringin (3, 36). The possible
might be a storage and transport form of monolignols (3, 36), and
that the UDPG:coniferyl alcohol glucosyltransferase, together
with coniferin-β-glucosidase, which releases the sugar moiety from
glucoconjugates in the cell wall, may regulate the storage and
mobilization of monolignols for lignin biosynthesis (37).
Arabidopsis accumulates monolignol glucosides in the cells of
its root and leaf tissues (32, 33). The vacuolar membrane vesicles
prepared from Arabidopsis rosette leaves displayed considerable
activity in sequestering coniferin and syringin in the presence of
ATP (Fig. 3; Table 1). In contrast, the plasma membrane vesicles
were inactive to the glucoconjugated monolignols in either the
presence or absence of ATP (Table 1). These data suggest that
glucosylation of monolignols is a prerequisite for their vacuolar
storage but not for the direct transport into cell walls of Arabi-
dopsis. These results complement previous genetic studies
wherein down- or up-regulating the expression of its UDPG:
monolignol glucosyltransferases entailed the corresponding re-
duction or accumulation of the soluble monolignol glucosides in
transgenic roots or leaves (32), but a change in lignin content or
composition was not observed.
The different chemical forms of monolignols required in ATP-
dependent transport also implicate the distinct classes of ABC
transporters involved in diverting and partitioning the polarized
“storage-form” glucosides of monolignols into vacuoles, and the
hydrophobic aglycones across plasmalemma.
Do Other Multidrug Membrane Transporters Participate in Monolignol
reportedly are involved in transporting a range of small secondary
metabolites, including phenolics and polyphenolics (20, 38). For
instance,themaize ABCtransporterZmMRP3, encoding aGS-X
pump and localized in the tonoplast, was shown, genetically, to be
necessary for translocating anthocyanin (39). However, seques-
related to anthocyanin, requires H+-gradient–dependent trans-
port (31, 40). The Arabidopsis mutant transparent testa12 (tt12),
lackingthe geneencoding a MATE-family secondary transporter-
like protein, exhibited much less deposition of proanthocyanidins
Transport activity (%)
coniferyl alcohol by inhibitors of membrane transporters. Inside-out vesicles
were incubated with 100 μM coniferyl alcohol, to which we added different
inhibitors at varied concentrations as described in Materials and Methods.
The average transport activity under MgATP and monolignol only was set
to 100%. The data are the means and SD of three replicates. *Significant
changes at P < 0.01 and **significant changes at P < 0.05, compared with
the control, under Student’s t test.
Inhibition of ATP-dependent uptake of plasma membrane vesicles to
Uptake of different phenolics by plasma and vacuolar
Plasma membraneVacuolar membrane
(nmol/mg protein) (10 min)
7.9 ± 2.0
3.0 ± 0.9
5.8 ± 1.3
1.7 ± 0.6
2.2 ± 0.2
1.1 ± 0.3
1.8 ± 0.3
0. 5 ± 0.1
0.6 ± 0.1
3.2 ± 0.9
0.2 ± 0.05
2.1 ± 0.2
0.6 ± 0.1
0.6 ± 0.1
0.2 ± 0.01
0. 7 ± 0.1
Uptake was measured in standard uptake medium containing various
monolignols in the presence or absence of 5 mM MgATP. Values shown
are mean ± SD (n = 3). ND, not detectable.
Miao and LiuPNAS
| December 28, 2010
| vol. 107
| no. 52
in the vacuoles of endothelial cells (41). Adding ABC transporter
inhibitors or blockers in uptake assays of plasma and vacuolar
membrane vesicles severely disrupted the uptake of monolignols
or their glucosylated derivatives (Figs. 2 and 3). However, iono-
phoric agents, such as gramicidin D, nigericin, and NH4Cl
which disturb the activities of MATE-family transporters, had
negligible effects on monolignol uptake (Fig. 2). These data im-
ply that the secondary energized MATE transporters may not
play vital roles as do the ABC transporters in mediating lignin-
Materials and Methods
Plant Material and Growth Conditions. Arabidopsis thaliana ecotype Colum-
bia (Col-0) was used in this study. For aerial tissues, seedling plants were
grown in soil for 4 wk with a controlled environment of 16/8-h light/dark
cycle (light, 100 μmol photons m−2·s−1, 22 °C; dark, 17 °C). Then, the plants
were harvested and stored at −80 °C until isolation of the microsomal
fractions. Populus tremuloides root tissues were collected from 8-mo-old
plants grown in the growth chamber under the same conditions.
Chemicals. All of the chemicals used were purchased from Sigma-Aldrich,
unless otherwise stated.
Preparation of Plasma Membranes and Formation of Inside-Out Vesicles. The
microsomal fractions from Arabidopsis rosette leaves and Populus root tis-
sues were prepared essentially as described by Palmgren et al. (24). The
resulting microsomes were suspended in 5 mM potassium phosphate buffer
(pH 7.8) containing 330 mM sucrose, 5 mM KCl, 1 mM DTT, and 0.1 mM EDTA
(buffer A). Plasma membranes (predominantly right-side-out vesicles) were
purified from the microsomal preparation by partitioning in an aqueous
polymer two-phase system, as described by Larsson et al. (42). Briefly, the
microsomal fraction was added to a phase system with a final weight of 36 g
and a final composition of 6.5% (wt/wt) dextran T500 and 6.5% (wt/wt)
polyethylene glycol 3350 in buffer A (pH 7.8), and then mixed and parti-
tioned. The final upper phase containing the right-side-out plasma mem-
branes was diluted severalfold with buffer A. The plasma membranes were
pelleted and resuspended to 15–20 mg/mL protein in the same buffer, and
stored at −80 °C until further use.
The purified right-side-out plasma membrane vesicles in buffer A were
mixed with the detergent Brij 58 (Sigma) to a final concentration of 0.05%
thawed in water at 20 °C four times to produce the sealed, inside-out vesi-
cles as described by Johansson et al. (25). The inverted plasma membranes
were pelleted at 100,000 × g for 2 h. The pellets were gently resuspended in
10 mM Mops·KOH buffer (buffer B; pH 7.5) containing 0.33 M sucrose, 0.1
mM EDTA, 1 mM DTT, and 1× protease inhibitor mixture (Sigma). We
monitored the quality of the prepared plasma membranes by measuring the
vanadate-inhibited Mg2+-ATPase activity with an ATPase assay kit according
to the manufacturer’s instructions (Innova Biosciences).
Preparation of Vacuolar Membrane Vesicles. Vacuolar membrane vesicles were
extracted from the remaining lower phase of the aqueous polymer two-
phase system (see above). They were diluted about 10-fold in buffer A, and
then pelleted at 100,000 × g for 2 h. All procedures were performed at 4 °C.
The pellet was resuspended in buffer A containing 1× protease inhibitor
1 / [S]
1 / V
1 / V
1 / [S]
Transport activity (% )
Absorbance at 280 nm (mAU)
(pmol / mg protein / min)
(pmol / mg protein / min)
uolar sequestration assay. (A) Protein gel-blot analysis
of selected fractions probed with antibodies against
V-PPase, Bip, and plasma membrane H+-ATPase. (B)
Portions of HPLC traces showing recovered coniferyl
alcohol 4-O-glucoside (coniferin) from the vacuolar
vesicles in the sequestering assay in the presence (a)
and absence (b) of ATP, the presence of sodium van-
adate and ATP (c), and the presence of gramicidin D
and ATP (d). (C) Effects of the membrane transporter
inhibitors sodium vanadate (1 mM) and gramicidin D
(5 μM) on the ATP-dependent uptake of coniferin by
vacuolar vesicles. Data were from three replicates.
*Significant changes at P < 0.01 under t test. (D and E)
The kinetics of transport of coniferin into vacuolar
vesicles for coniferin (D) and MgATP (E) concen-
trations. The data are from two or three replicates and
are plotted by nonlinear regression analysis fit to the
Michaelis–Menten equation. Insets show Lineweaver–
Preparation of vacuolar membranes and vac-
| www.pnas.org/cgi/doi/10.1073/pnas.1007747108Miao and Liu
mixture. The suspension was layered over a discontinuous sucrose gradient Download full-text
[10%, 15%, 20%, 25%, 30%, 40%, and 50% (wt/vol) in 20 mM Tris·HCl
buffer (pH 7.6), 1 mM DTT, and 1 mM EDTA] in a 40-mL tube. The tubes were
centrifuged at 100,000 × g for 3 h. Successive 2-mL fractions were collected
from the top of the centrifuge tube, diluted with buffer B, and again
centrifuged at 100,000 × g for another 2 h. The pellets were resuspended in
0.2 mL of buffer B for subsequent assays.
Measurement of Vacuolar ATPase and PPase Activity. Vacuolar ATPase activity
was measured by the method of Ames (43). PPi-dependent H+translocation
by vacuolar membrane vesicles was assayed fluorimetrically at 25 °C using
quinacrine as ΔpH indicator. For details, see SI Materials and Methods.
Transport Activity Assay. For the uptake assay, we modified a method de-
scribed by Zhao and Dixon (31) and Sugiyama et al. (27). The 500-μL assay
mixtures contained 25 mM Tris·Mes (pH 8.0), 0.4 M sorbitol, 50 mM KCl, 5
mM MgATP, 0.1% (wt/vol) BSA, and the indicated concentration of phenolic
substrate. ATP was omitted from the nonenergized controls. Assays were
started by adding the membrane vesicles (50–100 μg of protein) while briefly
agitating the mixture at 25 °C. Batches of the reaction mixture (100 μL) were
removed at various times, and their reactions were terminated with 1.0 mL
of ice-cold washing solution (25 mM Tris·Mes, pH 8.0, 0.4 M sorbitol). The
mixtures underwent vacuum filtration through prewetted nitrocellulose
membrane filters (0.22-μm pore diameter; Millipore). The dried filters,
transferred to 20-mL glass vials containing 0.5 mL of 50% (vol/vol) methanol,
were extracted for 1 h at room temperature in an orbital shaker. The eluate
was analyzed by HPLC. The sample was resolved on a Gemini C18 reverse-
phase column (Phenomenex) in 0.2% acetic acid (A) with an increasing
concentration gradient of acetonitrile containing 0.2% acetic acid (B):
0–20 min, 30% B; 20–25 min, 100% B at a constant rate of 0.8 mL/min.
UV absorption was monitored at 254, 280, and 310 nm using a multiple-
wavelength photodiode array detector (Agilent).
For our tests of transport inhibitors, we preincubated them with the
membrane vesicles for 2 min at the following final concentrations: 1 mM
vanadate (in water), 5 μM verapamil (in DMSO), 150 μM glybenclamide (in
DMSO), 5 μM gramicidin D (in DMSO), 2 μM nigericin (in DMSO), 50 μM ni-
fedipine (in DMSO), 1 mM potassium cyanide (in water), and 1 mM NH4Cl (in
water). Sodium vanadate was depolymerized before use according to Good-
effects, each inhibitor was used at the concentration given for its specific in-
hibitory effect on transporters and pumps, or for disrupting membrane po-
tential as well as ΔpH based on values in the literature. To the 250-μL assay
mixture, 1–2.5 μL of each stock solution was added, where the concentration
of organic solvent was less than 1% (vol/vol). After incubation at 25 °C for 30
min, the transported phenolics were measured as described above. The sta-
tistical analyses on the obtained datasets were carried out with a Student’s
Kinetics of Transport of Monolignol or Its Glucoside. We assessed the kinetics
of transport of coniferyl alcohol by plasma membrane vesicles or of coniferin
by vacuolar vesicles. For details, see SI Materials and Methods.
ACKNOWLEDGMENTS. This work was supported by the Division of Chemical
Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the
US Department of Energy (DOE) through Grant DEAC0298CH10886 to C.-J.L.
Initially, this work was also partially supported by the Office of Biological
and Environmental Research of DOE through the pilot project of biofuel
Scientific Focus Area program (BO148).
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Miao and Liu PNAS
| December 28, 2010
| vol. 107
| no. 52