Download Post-transcriptional regulation of auxin transport proteins: cellular

Journal of Experimental Botany, Vol. 60, No. 4, pp. 1093–1107, 2009
doi:10.1093/jxb/ern240 Advance Access publication 29 September, 2008
REVIEW PAPER
Post-transcriptional regulation of auxin transport proteins:
cellular trafficking, protein phosphorylation, protein
maturation, ubiquitination, and membrane composition
Boosaree Titapiwatanakun and Angus S. Murphy*
Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-2010, USA
Received 2 July 2008; Revised 28 August 2008; Accepted 1 September 2008
Abstract
Auxin concentration gradients, established by polar transport of auxin, are essential for the establishment and
maintenance of polar growth and morphological patterning. Three families of cellular transport proteins, PIN-formed
(PIN), P-glycoprotein (ABCB/PGP), and AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX), can independently and coordinately transport auxin in plants. Regulation of these proteins involves intricate and co-ordinated cellular
processes, including protein–protein interactions, vesicular trafficking, protein phosphorylation, ubiquitination, and
stabilization of the transporter complexes on the plasma membrane.
Key words: AUX/LAX, cellular trafficking, membrane composition, P-glycoprotein, phosphorylation, pin-formed, protein
maturation, ubiquitination.
Introduction
The phytohormone indole-3-acetic acid (IAA), or auxin,
plays an essential role in embryogenesis (Friml et al., 2003),
cell division and elongation (Campanoni and Nick, 2005),
vascular tissue differentiation (Mattsson et al., 2003),
phyllotactic patterning (Bainbridge et al., 2008), lateral root
formation (Dubrovsky et al., 2008), phototropism (Kimura
and Kagawa, 2006), gravitropism (Palme et al., 2006), and
other physiological processes. Although auxin was the first
phytohormone to be discovered (Went, 1927), the molecular
mechanisms underlying its transport and perception have
only been elucidated in the past decade (Kepinski, 2007;
Delker et al., 2008).
IAA is synthesized in the shoot, particularly by leaf
primordia and young leaves, and transported to the root
through vascular and bundle sheath tissues (Ljung et al.,
2005; Bandyopadhyay et al., 2007). The synthesis, transport, and catabolism of IAA is tightly regulated by both
transcriptional and post-transcriptional processes that are
co-ordinately regulated via the ubiquitination of AUX/
IAA repressor proteins by the SCFTIR1/AFB mechanism
followed by proteolytic degradation (Quint and Gray,
2006). Additional post-transcriptional mechanisms further
regulate auxin transport. This review focuses on the role of
post-translational mechanisms that regulate auxin transport processes by modifying, activating, redistributing, or
degrading auxin transport proteins or protein complexes.
Polar auxin transport
Auxin is polarly transported from cell to cell in a process
that involves chemiosmotically-driven export to and uptake from the apoplast. As IAA is a weak organic acid
(pKa¼4.75), it exhibits a ;5:1 distribution of anionic
(IAA–): protonated (IAAH) speciation in the acidic (pH
;5.5) apoplast. IAA can thus enter the cell by either lipophilic diffusion of IAAH or by anionic uptake via H+IAA– symporters. The latter process is required as the
diffusive flux of IAAH across the plasma membrane is
thought to be an order of magnitude slower than that of
carrier-mediated IAA– translocation, and diffusion alone
cannot account for auxin fluxes that naturally occur in
plants (Kramer and Bennett, 2006). In contrast, the
* To whom correspondence should be addressed: E-mail: [email protected]
ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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1094 | Titapiwatanakun and Murphy
cellular efflux of IAA requires protein mediation, as IAA is
almost exclusively anionic in the cytoplasm (pH ;7.0) and
cannot diffuse across the membrane on its own. The localization and activity of auxin transport complexes are
thus crucial in establishing the polarity of auxin transport.
The concentration gradient created by directional movement of auxin is fundamental to the establishment of plant
axial polarity, organ patterning, and morphological adaptation to the environment (De Smet and Jurgens, 2007). From
the first cell division in plant embryogenesis, auxin is
preferentially accumulated in the zygotic apical cell where it
functions as an important determinant of that cell’s proembryonic fate (Friml et al., 2003). Vascular differentiation
also coincides with auxin accumulation in preprocambial
cells (Mattsson et al., 2003). In the root, acropetal transport
(base to apex) of auxin (Blakeslee et al., 2005a) within the
stele is responsible for the initiation of lateral root
primordia from pericycle cells that can respond to auxin
activation (Casimiro et al., 2001; Bhalerao et al., 2002; Wu
et al., 2007). In Arabidopsis, the priming of pericycle
cells for auxin responsiveness occurs in the basal region of
the root meristem and is controlled by a periodic shift in the
basipetal (apex to base) redistribution of auxin through the
lateral root cap and epidermal cell files, resulting in an
alternating pattern of regularly spaced lateral roots (De
Smet et al., 2007). In gravitropic root bending, asymmetric
changes in the basipetal (root apex to base) transport
stream caused by alteration in the gravitational vector leads
to differential root elongation and bending in the direction
of the gravitational vector (Palme et al., 2006). Similarly,
phototropic bending in hypocotyls is thought to result from
asymmetric accumulation of auxin in cells distal to the site
of illumination resulting in asymmetric growth and bending
of the hypocotyl toward light (Kimura and Kagawa, 2006).
Classes of auxin transport proteins
Auxin transport proteins have been identified and, to date,
have been grouped in three families: AUXIN RESISTANT
1/LIKE AUX1 (AUX1/LAX) uptake symporters, PINFORMED (PIN) efflux carriers, and P-GLYCOPROTEIN
(MDR/PGP/ABCB) efflux/conditional transporters.
AUX1/LAX uptake symporters
AUX1 was originally identified in a genetic screen for
Arabidopsis mutants that exhibited auxin-resistant root
growth (Bennett et al., 1996). The AUX1 gene encodes a
transmembrane protein similar to amino acid permeases.
AUX1 participates in loading of shoot auxin into the
phloem for long-distance transport toward the root tip and
in the basipetal transport of auxin out of the lateral root
cap at the root apex (Swarup et al., 2002, 2004). The ability
of AUX1 to mediate auxin influx has been demonstrated in
planta and by expression in heterologous systems. Treatment with the membrane-permeable artificial auxin 1naphthaleneacetic acid (1-NAA) was shown to rescue the
agravitropic phenotype of the aux1 mutant, which is also
resistant to the weakly permeate and poorly transported
auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)
(Marchant et al., 1999). When expressed in Xenopus
oocytes, AUX1 was shown to function as a high-affinity
auxin uptake carrier protein (Yang et al., 2006; Kerr and
Bennett, 2007).
AUX1 is localized to the lower end of the cells in the
lateral root cap, epidermal cells below the elongation zone,
columella, and protophloem (Kleine-Vehn et al., 2006). The
three members of the Like-AUX1 (LAX) family are functional analogues of AUX1, and appear to function in tissuespecific auxin uptake (Swarup et al., 2004). Quadruple aux/
lax mutants exhibit aberrant formation of leaf primordia
and reduced polar PIN localization consistent with altered
auxin flux (Bainbridge et al., 2008). LAX3 functions in the
early stages of lateral root formation (de Billy et al., 2001;
Swarup et al., 2008).
PIN efflux carriers
The Arabidopsis pin-formed1 (pin1) mutant exhibits defects
in vascular patterning, organogenesis, and phyllotaxis
(Galweiler et al., 1998; Reinhardt, 2005). PIN proteins
belong to the unique auxin efflux facilitator family found in
plants and some fungi and are predicted to have 10–12
transmembrane domains (Galweiler et al., 1998; Blakeslee
et al., 2007). Among the eight members of the PIN family in
Arabidopsis, five have been experimentally shown to function as auxin efflux carrier proteins when expressed in
Arabidopsis, tobacco BY-2, human HeLa, and/or yeast cell
cultures (Petrasek et al., 2006; Blakeslee et al., 2007). PINmediated efflux in these heterologous systems was partially
inhibited by the auxin efflux inhibitor 1-naphthylphthalamic
acid (NPA), although, in all cases when it was used, NPA
strongly inhibited background auxin efflux in cells not
expressing recombinant PIN proteins, especially in plant
cell systems (Petrasek et al., 2006).
Subcellular localization of PIN proteins maps with the
directionality of auxin transport vectors, especially in
embryonic development and organogenesis (Benkova et al.,
2003; Blilou et al., 2005). In the central vascular cylinder,
PIN1 is basally localized in the xylem parenchyma and participates in the transport of auxin along the embryonic axis
from the shoot to the root tip. PIN2 exhibits a basal
(bottom) and lateral localization in root cortical cells and
apical (top) localization in root epidermal cells consistent
with its apparent role in redirection and reflux of auxin at
the root tip (Chen et al., 1998; Luschnig et al., 1998; Muller
et al., 1998). PIN3 exhibits an apolar orientation in root
columella cells, but relocalizes in the direction of auxin
movement upon gravistimulation (Friml et al., 2002b). PIN3
is also localized to the inner surface of hypocotyl bundle
sheath cells where it appears to function in the redirection
of auxin back into the vascular cylinder. PIN3 is also found
in epidermal cells, and the pin3 mutant shows shorter epidermal cells in light-grown hypocotyls, thought to be caused
by a defect in cell elongation (Friml et al., 2002b). As
Post-transcriptional regulation of auxin transport proteins | 1095
mutational analysis indicates that PIN3 functions in tropic
responses, activation of its transport activity near sites of
illumination could be expected to accelerate auxin movement out of tissues on the illuminated (non-bending side) of
the hypocotyl.
Other PIN proteins exhibit primarily apolar subcellular
localizations but still contribute to directional auxin movement. PIN4 exhibits mixed polar and apolar localizations in
the provascular quiescent centre and daughter cells and
functions in root meristem patterning (Friml et al., 2002a).
PIN7 is abundant in epidermal tissues where it exhibits a
non-polar localization (Blakeslee et al., 2007), and is also
involved in the establishment of the apical–basal axis,
particularly in the hypophysis (Friml et al., 2003). Despite
their seemingly discrete expression patterns and functions,
some redundancy is found among PIN family members as
compensatory expression of some PIN genes was observed
in pin1 and pin2 mutants, and ectopic expression of these
PIN homologues was sufficient to rescue the auxin transport phenotype of the mutants (Vieten et al., 2005). One
member of the PIN family, PIN5, is particularly intriguing,
as it lacks a variable central domain common to other characterized PIN proteins that, in the case of PIN1 has been
shown to mediate protein–protein interactions (Blakeslee
et al., 2007). Analysis of PIN5 auxin transport activity (if
any) will help determine whether the central variable
domain of the PIN proteins plays a functional role in
transport or has a primarily regulatory function as has been
proposed.
Some evidence suggests that auxin efflux proteins also
mediate intercellular auxin movement from mildly alkaline
organelles to the neutral cytosol. Anionic auxin accumulation within compartments with a pH >7.0 is predicted by
chemiosmotic models, and the essential auxin binding
protein ABP1 is relatively abundant in the mildly alkaline
endoplasmic reticulum (Timpte, 2001). Further, the toxic
effects of the artificial auxin 2,4-dichlorophenoxyacetic acid
(2,4-D) are associated with its accumulation in the endoplasmic reticulum (Dharmasiri et al., 2006). As compared to
IAA, 2,4-D is poorly transported by efflux carrier proteins,
one of the uncharacterized PIN transporters, such as PIN5
or PIN8, and/or one or more uncharacterized ABCB
transporter may mediate auxin efflux from the ER.
ABCB efflux transporters
A third class of auxin transporters are phospho-glycoproteins
(PGPs) that belong to the ABCB subgroup of the ATPBinding-Cassette (ABC) transporter superfamily. The best
known member of the ABCB subgroup is the human
ABCB1 protein which has been extensively studied for its
role in increased resistance to chemotherapeutic agents
resulting from its overexpression in cancer cells (Luckie
et al., 2003). However, the use of the multidrug resistance
(MDR) nomenclature for this subgroup of proteins has
been discontinued as the majority of family members appear to exhibit a higher degree of transport substrate
specificity than mammalian ABCB1 (Verrier et al., 2008).
In Arabidopsis thaliana, the 21 members of ABCB subgroup
exhibit both distinct and overlapping expression patterns
throughout all stages of plant growth and development
(Blakeslee et al., 2005b). The best characterized members of
Arabidopsis ABCB proteins are the auxin transporters
ABCB1, ABCB4, and ABCB19. Multiple reports have
catalogued PIN and AUX/LAX gene expression and protein localization (Blakeslee et al., 2005a, b; Tanaka et al.,
2006; Zazimalova et al., 2007). By contrast, a current
summary of ABCB auxin transporter gene expression and
ABCB protein distribution is lacking in the literature. A
brief summary of the expression patterns of ABCB auxin
transporter genes is provided in Table 1 and a summary of
protein localization is shown in Fig. 1.
The involvement of ABCB proteins in auxin transport
was first suggested by Sidler et al., (1998) when expression
levels of PGP1/ABCB1 in Arabidopsis were found to
regulate hypocotyl elongation in a light-dependent manner
(Sidler et al., 1998). ABCB1 was subsequently shown to
function co-ordinately with PGP19/MDR1/ABCB19 in
mediating polar auxin transport in Arabidopsis (Noh et al.,
2001). The sequence of ABCB19 is highly similar to that of
ABCB1 (Verrier et al., 2008). Arabidopsis abcb1 and abcb19
mutants exhibit reductions in both growth and root
basipetal auxin transport with the most pronounced reductions seen in the double abcb1 abcb19 mutant. Polar auxin
transport is reduced ;70% in abcb1 abcb19, while pin1
exhibits a ;30% reduction (Blakeslee et al., 2007), but abcb
mutants show none of the defects in organogenesis that are
seen in pin1 (Noh et al., 2001). This suggests that ABCBs
primarily regulate long-distance auxin transport and localized loading of auxin into the transport system and do not
function in establishing the basal vectorial auxin flows that
function in organogenesis (Bandyopadhyay et al., 2007;
Blakeslee et al., 2007; Bailly et al., 2008). This interpretation
of ABCB auxin transport function was confirmed when loss
of function of ABCB1 orthologues were found to underlie
the dwarf phenotypes of the agriculturally-important brachytic2/zmabcb1 maize and dwarf3/sbabcb1 sorghum
mutants (Multani et al., 2003).
Arabidopsis abcb19 also exhibits exaggerated phototropic
and gravitropic responses (Noh et al., 2001, 2003; Lin and
Wang, 2005; Lewis et al., 2007; Wu et al., 2007). In
addition, it has recently been shown that the expression
level of ABCB19 is suppressed upon activation of the
phytochrome and cryptochrome photoreceptors in response
to the red and blue light, respectively (Nagashima et al.,
2008). These results point to an ABCB19 function in the
repression of the differential growth of the light- and
gravity-stimulated hypocotyl (Noh et al., 2003; Nagashima
et al., 2008).
A direct role for ABCB1 and ABCB19 in cellular efflux
was demonstrated when increased auxin retention was
observed in mesophyll protoplasts from Arabidopsis abcb1
and abcb19 mutants (Geisler et al., 2005). Further, as was
seen with PIN proteins, heterologous expression of
ABCB1 and ABCB19 in HeLa and/or yeast cells resulted
in enhanced auxin efflux that was inhibited by NPA
1096 | Titapiwatanakun and Murphy
Table 1. Summary of microarray data indicating ABCB1, ABCB4, and ABCB19 gene expression (from Genevestigator, https://
www.genevestigator.ethz.ch/)
The highest expression is shown in bold. Data presented are means and standard errors of normalized Affymetrix expression values.
Anatomy
ABCB1
ABCB4
Mean
0 Callus
1 Cell suspension
2 Seedling
21 Cotyledons
22 Hypocotyl
23 Radicle
3 Inflorescence
31 Flower
311 Carpel
3111 Ovary
3112 Stigma
312 Petal
313 Sepal
314 Stamen
3141 Pollen
315 Pedicel
32 Silique
33 Seed
34 Stem
35 Node
36 Shoot apex
37 Cauline leaf
4 Rosette
41 Juvenile leaf
42 Adult leaf
43 Petiole
44 Senescent leaf
45 Hypocotyl
451 Xylem
452 Cork
5 Roots
52 Lateral root
53 Root tip
54 Elongation zone
55 Root hair zone
56 Endodermis
57 Endodermis+cortex
58 Epid. atrichoblasts
59 Lateral root cap
60 Stele
5538
5270
2889
2186
5951
4265
4538
4562
5241
3926
7374
4601
3231
1256
53
8765
5351
1641
8620
10654
5026
2588
2497
2492
1999
3512
1609
3250
2667
4544
4245
4685
2913
4845
5197
4094
3338
4458
4985
4351
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
(Geisler et al., 2005; Bouchard et al., 2006). However,
unlike what was seen with PIN expression, the efflux
mediated by the ABCBs in mammalian cells was insensitive to inhibitors of mammalian organic anion transporters, suggesting that ABCB-mediated auxin export did
not involve activation of an endogenous transport activity
(Geisler et al., 2005; Petrasek et al., 2006).
ABCB4 functions in the movement of auxin away from
the root tip and appears to function primarily in the regulated export of auxin out of the elongation zone (Santelia
SE
Mean
406
294
53
163
922
265
185
235
449
157
642
698
331
321
9
134
554
108
508
134
229
141
41
130
50
421
62
252
74
215
81
392
191
179
563
774
146
413
344
98
4427
3766
1646
186
1343
2324
366
144
50
57
42
46
883
44
27
139
202
885
502
475
100
540
708
1267
540
143
1138
2293
3225
1544
4187
2841
1791
4119
8427
4560
2266
2864
3151
2303
ABCB19
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
SE
Mean
368
256
72
24
330
92
46
25
7
8
12
14
113
12
14
18
40
174
76
21
9
105
58
335
43
15
69
296
93
119
183
803
361
696
1175
279
569
85
787
101
2037
807
2485
1786
1769
3111
2794
3348
4205
2223
619
5024
921
1561
274
4159
1889
1655
2068
1588
4827
747
1413
1253
957
4356
254
424
180
585
3197
1496
6225
5012
4868
5503
7226
3511
4154
5567
SE
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
132
107
50
227
381
106
134
231
822
264
105
1004
334
511
13
120
246
251
259
59
311
26
32
66
42
227
11
80
18
66
82
391
886
1084
555
571
148
415
469
435
et al., 2005; Terasaka et al., 2005; Cho et al., 2007; Lewis
et al., 2007). ABCB4 exhibits structural similarity to the
berberine uptake transporter CjMDR1/CjABCB1 from the
medicinal plant Coptis japonica (Shitan et al., 2003) and, to
a lesser extent, the ABCB14 malate uptake transporter from
Arabidopsis guard cells (Lee et al., 2008), but exhibits
dissimilarity in putative substrate binding sites (H Yang and
A Murphy, unpublished data). Some experimental evidence
indicates that ABCB4 functions in auxin uptake. When
expressed in mammalian cells, ABCB4 activates a net
Post-transcriptional regulation of auxin transport proteins | 1097
Fig. 1. Localization of ABCB1, ABCB4, and ABCB19 proteins in Arabidopsis roots. Shown are ProABCB1:ABCB1-GFP (transgenic lines
courtesy from Dr Jiri Friml), ProABCB4:ABCB4-GFP (transgenic lines courtesy from Dr Misuk Cho), and ProABCB19:ABCB19-GFP
(transgenic lines courtesy from Dr Jiri Friml). Bar¼25 lm.
increase in auxin retention (Terasaka et al., 2005), and
heterologous expression of ABCB4 in the yeast IAAsensitive yap1 mutant led to enhanced growth sensitivity to
IAA (Santelia et al., 2005). However, NPA treatment of
mammalian cells expressing ABCB4 activated efflux to
levels equivalent to that seen in NPA-treated cells expressing Arabidopsis ABCB1 or ABCB19 (Terasaka et al., 2005).
Recently, ABCB4 expression has been found to confer
auxin efflux activity in root hairs and tobacco suspension
cells (Cho et al., 2007). These results suggest that the
directionality of ABCB4-mediated transport is regulated by
plant-specific modulators (Cho et al., 2007). However, it is
also possible that ABCB4 export is directly activated by
threshold levels of transport substrates as is the case with
human ABCB1 (Kimura et al., 2007). An examination of
published results suggests that auxin efflux activated by
ABCB4 is more evident in experiments utilizing longer time
periods for assays, while activation of uptake is seen with
lower concentrations in shorter time periods. As such,
ABCB4 might best be referred to as a conditional auxin
efflux/uptake transporter. Structural modelling comparisons
of ABCB4 with ABCB1, ABCB19, and the ABCB14 malate
uptake transporter (H Yang and A Murphy, unpublished
results) suggest that unique structure/sequence variations in
ABCB4 underlie its conditional activity.
ABCB4 appears to function primarily in the accumulation of auxin in the elongation zone as well as efflux from
root trichoblast cells. Mutations in ABCB4 exhibit decreased
linear growth (Terasaka et al., 2005), increased initial rates of
root bending (Lewis et al., 2007), and altered root hair
formation (Santelia et al., 2005; Cho et al., 2007) that
are dependent on growth conditions. All of these functions
are consistent with ABCB4 expression patterns, ABCB4
protein distribution and subcellular localization, and auxin
transport profiles of abcb4 mutants.
Regulation of auxin transport proteins by
protein–protein interactions
Activation of ABCB proteins by TWD1/FKBP42
Multiple lines of evidence suggest that there are at least two
distinct NPA-binding sites in Arabidopsis membranes. A
high affinity site associated with the inhibition of auxin
transport at the plasma membrane is associated with an
integral membrane protein, and possibly, an associated
peripheral protein (Murphy et al., 2002). A second, low
affinity NPA binding site is thought to be a membrane anchored or peripheral amidase (Murphy et al., 2002). Other
sites of NPA action have been associated with membrane
trafficking events, but require such high concentrations of
NPA to be visualized that they can be regarded as nonspecific (Geldner et al., 2001). NPA affinity chromatography was initially used to isolate the ABCB1, 4, and 19
proteins (Murphy et al., 2002; Geisler et al., 2003; Terasaka
et al., 2005). An FKBP immunophilin-like protein, TWD1/
FKBP42 was copurified with the ABCBs (Murphy et al.,
2002).
Subsequent studies have established that the C-terminal
domains of ABCB1 and ABCB19 interact with FKBP42
and that the phenotypes of abcb1 abcb19 resemble those of
the twd1 mutant (Geisler et al., 2003). Based on sequence
prediction, FKBP42 was proposed to be a glycophosphatidyl inositol (GPI)-anchored protein (Geisler et al., 2003).
However, no GPI moiety was detected when TWD1 was
biochemically analysed (Murphy et al., 2002; Granzin et al.,
2006), and subsequent structural characterizations are inconsistent with the presence of a GPI anchor (Eckhoff et al.,
2005). Further, the abundance of FKBP42 is very low
compared to ABCB1 and ABCB19 (Bailly et al., 2008),
suggesting that FKBP42 functions in activating ABCB
membrane complexes, rather than anchoring complex
1098 | Titapiwatanakun and Murphy
formation. FKBP42 has been proposed to induce conformational changes in ABCB1 and ABCB19 (Geisler et al.,
2003; Bouchard et al., 2006; Bailly et al., 2008). ABCB1–
FKBP42 interactions have been shown to be sensitive to
both NPA and flavonoid inhibitors of auxin transport,
which appear to interact with multiple sites of action in the
respective proteins (Bailly et al., 2008). Conformational
changes are involved in the regulation of mammalian ABCB
(Ambudkar et al., 2006), although little is known about the
protein interactions that initiate these changes.
Consistent with this proposed function, loss of FKBP42
conferred resistance to the pharmacological agent gravacin,
which is an inhibitor of ABCB19 activity (Rojas-Pierce
et al., 2007). Gravacin was originally identified as an
inhibitor of gravitropic bending in hypocotyls and was
subsequently found to interfere with subcellular targeting of
vacuolar marker proteins (Surpin et al., 2005). Wild-type
Arabidopsis seedlings treated with gravacin resulted in
reductions of auxin transport that were similar to those
seen in abcb19, while gravacin treatment of abcb19 mutants
resulted in nominal further reductions in auxin transport
(Rojas-Pierce et al., 2007). A screen for mutants that are
resistant to gravacin resulted in the identification of abcb/
pgp19-4 which harbours a point mutation in the C-terminal
domain of ABCB19 (E1174K) (Rojas-Pierce et al., 2007).
Treatment with gravacin did not alter the gravitropic
response of twd1, and microsomes derived from twd1
showed reduced binding to gravacin (Rojas-Pierce et al.,
2007; Bailly et al., 2008). However, the immunolocalization
of ABCB19 was unchanged in twd1 mutants compared to
wild type (Titapiwatanakun et al., 2008), suggesting that
FKBP42 is more likely to function in activation, rather than
localization of ABCB1/19 to the plasma membrane.
PIN-ABCB interactions
Although both ABCB19 and PIN1 can function as independent auxin efflux transporters, these proteins can interact and co-ordinately transport auxin (Bandyopadhyay
et al., 2007; Blakeslee et al., 2007). Co-ordinated, but
independent functions for PINs and ABCB1/19 are particularly evident in embryonic development and lateral root
formation. However, physical interaction between ABCB19
and PIN1 in post-embryonic growth is suggested by positive
results in subcellular colocalization, coimmunoprecipitation, and yeast two-hybrid interaction analyses (Blakeslee
et al., 2007). Moreover, functional interactions between
ABCB19 and PIN1 are supported by apparently synergistic
phenotypes of pin1 abcb19 mutants and enhanced auxin
efflux activity, inhibitor sensitivity, and substrate specificity
of HeLa cells co-expressing ABCB19 and PIN1. As was the
case with FKBP42/TWD1, interactions of ABCB19 with
PIN1 are mediated by the C-terminal domain of the
protein, and gravacin effectively interferes with the enhanced auxin transport mediated by ABCB19 and PIN1
coexpression (Rojas-Pierce et al., 2007).
Some evidence of interactions between PIN1 and ABCB1
are suggested by co-immunoprecipitation studies and by
increases in auxin efflux when the two proteins are coexpressed in heterologous systems (Blakeslee et al., 2007).
However, PIN1–ABCB1 interactions appear to be indirect,
as no evidence of protein interactions are seen in yeast twohybrid assays (Blakeslee et al., 2007). By contrast, interactions of PIN2 with ABCB1 may be more robust, as
coexpression of PIN2 and ABCB1 in yeast has synergistic
effects on auxin transport and pin2 pgp1 pgp19 mutants
exhibit severely agravitropic growth phenotypes (Blakeslee
et al., 2007).
Regulation of auxin transporters by auxin
levels and fluxes
Auxin has been proposed to ‘canalize’ it own transport by
reorienting transport components (Sachs, 1981), presumably by altering the subcellular localization of auxin transport proteins or by regulating their transport activity. The
direction of auxin flow has been shown to influence polar
PIN localization in a cell type-specific manner (Sauer et al.,
2006). For instance, in graviresponding root columella cells,
PIN3 is disproportionately oriented on the lower side of
cells in the path of auxin destined to accumulate in the
epidermal cells of the distal elongation zone (Friml et al.,
2002b). Similarly, expression of an apically-localized chimeric PIN1 protein under the control of the PIN2 promoter
was able to suppress the root agravitropic phenotype of the
pin2 mutant, while comparable localization of basallylocalized PIN1 protein failed to produce the same result
(Wisniewska et al., 2006). To date, most studies have
focused on easily visualized changes in PIN protein
localization in response to altered auxin fluxes. However, it
is unlikely that PIN proteins are only regulated by localized
auxin accumulations. Numerous studies have shown that
PIN expression and PIN protein abundance are altered by
changes in auxin levels or transport (Peer et al., 2004;
Vieten et al., 2005). It is likely that protein phosphorylation
and turnover of PINs play an important role in auxindependent regulation of PIN function as well.
Expression of ABCB1, ABCB4, and ABCB19 is upregulated by auxin application (Noh et al., 2001; Geisler
et al., 2005; Terasaka et al., 2005). The promoter sequence
of ABCB1 includes auxin responsive motifs and the timecourse of b-glucuronidase (GUS) expression directed by
ABCB1 promoter (ABCB1pro:GUS) at the shoot and root
apices in response to auxin treatment indicate that ABCB1
expression responds rapidly to auxin treatment (Geisler
et al., 2005). By contrast, ABCB4 appears to be a ‘late’
auxin response gene (Terasaka et al., 2005). In general,
protein abundances of ABCB1, 4, and 19 appear to reflect
gene expression levels (Geisler et al., 2005; Terasaka et al.,
2005; Blakeslee et al., 2007; Wu et al., 2007). Overexpression of ABCB4 does not result in the presence of ectopic
ABCB4 protein in the shoot (Terasaka et al., 2005). This
appears to be a result of regulation of gene expression or
RNA stability, as steady-state ABCB4 RNA levels in the
shoot were found to be very low in overexpressing lines
Post-transcriptional regulation of auxin transport proteins | 1099
(Terasaka et al., 2005). A similar disparity is found with
expression of ABCB19, as visualized with a MDR1pro:
GFP-MDR1 fusion. No signal is seen in root and anther
filament epidermal cells where transcriptional reporters,
RT-PCR, and microarray expression reports indicate gene
expression (Blakeslee et al., 2007; Wu et al., 2007).
However, immunolocalizations, epitope-tags, and other
protein–reporter fusions indicate that the protein is present
in these cells (Blakeslee et al., 2007). Further, ABCB19
appears to be highly stable and protein abundance responds
slowly to decreases in gene expression (Titapiwatanakun
et al., 2008).
Regulation of auxin transporters by cellular
trafficking mechanisms
The trafficking pathways of the PIN and AUX1 auxin
transport proteins have been extensively investigated. Less
is known about the trafficking of the ABCBs, primarily due
to a strong community bias against the acceptance of these
proteins as auxin transporters until recently. In Fig. 2,
a summary of the known subcellular trafficking pathways
that mediate PIN1, PIN2, AUX1, ABCB1, and ABCB19 to
and from the plasma membrane is presented.
Trafficking mechanism of PINs
The dynamic trafficking of PIN1 is the one of the best
studied trafficking mechanisms in plants. PIN1 trafficking is
mediated by ADP-ribosylation factors (ARFs), the GNOM
ARF guanine nucleotide exchange factor (ARF-GEF)
(Steinmann et al., 1999), and the ARF GTPase activating
protein (ARF-GAP) SCARFACE (SFC) (Sieburth et al.,
2006). Embryos of gnom mutants exhibit severe polarity
defects and altered polar localizations of PIN1 (Steinmann
et al., 1999). The Sec7 domain of GNOM has been shown
to be a target of the fungal toxin brefeldin A (Geldner et al.,
2003), and short-term treatments of brefeldin A (BFA) have
been shown to reversibly cause intracellular aggregations of
PIN1 in cells at the root apex (Geldner et al., 2001).
Mutation of the Sec7 domain of GNOM to a BFAinsensitive form (GNOMM696L) prevents the formation of
PIN1 aggregations after BFA treatment (Geldner et al.,
2003), indicating that PIN1 trafficking is mediated by
GNOM. PIN3 has also been shown to associate with the
BFA compartment following BFA treatment (Friml et al.,
2002b). PIN1 accumulation after BFA treatment in the sfc
mutant resulted in multiple smaller organelles, and implicated a role of the SCARFACE ARF-GAP protein in PIN1
cycling as well (Sieburth et al., 2006). In addition, trafficking of PIN1 also requires an intact actin network, but
appears not to require microtubules (Geldner et al., 2001).
The time- and dosage-dependent effects of BFA on PIN
trafficking have recently been examined (Kleine-Vehn et al.,
2008). Long-term incubation with BFA has been shown to
promote transcytosis of PIN1 and PIN2, in which the ARFGEF-mediated endocytotic vesicles was found to be translocated from one side of the cell to the other.
Fig. 2. Model of cellular trafficking pathways utilized by the auxin
transporter proteins. Movement of auxin depends on the pH
difference between the apoplast and the cytoplasm. The cellular
uptake of auxin occurs via the diffusion of IAAH and the import of
IAA– through the proton-symporter protein AUX1 (purple). The
passage out of the cell of IAA– is characterized by PIN-formed
proteins, shown here for PIN1 (blue) and PIN2 (green). An ATPdependent transporter, ABCB proteins (pink), are also mediated by
cellular auxin efflux. Although the trafficking of both PIN1 and PIN2
is sensitive to BFA, only PIN1 has been shown to utilize the
GNOM-dependent pathway. The trafficking of PIN2 depends on
the SNX1- and VPS29-containing retromer complex, which is
inhibited by wortmannin. The intracellular pool of PIN2 is targeted
to proteolysis though ubiquitination. AXR4, an ER-localized protein, is required for the plasma membrane localization of AUX1.
The trafficking pathways of AUX1 and ABCB1 are also sensitive to
BFA. The trafficking of ABCB19 is not regulated by the same
dynamic mechanisms used by PIN1 and PIN2. Gravacin inhibits
the targeting of ABCB19 to the plasma membrane.
The subcellular trafficking of PIN2 appears to be
regulated by different mechanisms than those mobilizing
PIN1. Trafficking of PIN2 is mediated by endosomes
containing SORTING NEXIN1 (SNX1), which are distinct
from GNOM-mediated endosomes and are sensitive to the
phosphatidylinositol-3-OH kinase (PI-3K) inhibitor wortmannin (Jaillais et al., 2006). SNX1 is a subunit of the
retromer complex, which functions in recycling transmembrane proteins from endosomal multivesicular bodies (MVBs)
1100 | Titapiwatanakun and Murphy
to the trans-Golgi network in yeast and mammals (Bonifacino and Rojas, 2006). Another important subunit of the
retromer complex is the Vacuolar Protein Sorting 29
(VPS29), which is also localized to MVBs. Arabidopsis mutants lacking VPS29 showed the alterations in the morphology of SNX1-containing endosomes and also altered
PIN1 localization (Jaillais et al., 2007), suggesting that the
retromer complex functions in the endosomal recycling of
PIN1 as well.
The endocytosis of PIN proteins has been shown to be
inhibited by auxin itself (Paciorek et al., 2005; Zazimalova
et al., 2007). Treatment with high auxin concentrations stabilized PIN proteins (PIN1, 2, 3, and 4) on the
plasma membrane. This phenomenon may reflect a need for
increased PIN-mediated auxin efflux transport activity in
order to prevent accumulation of auxin in the cytoplasm.
However, there appears to be a difference between treatment with high concentrations of exogenous auxin and
manipulation of auxin transport itself. The application of
picomole amounts of auxin at the shoot apex to enhance
auxin flux artificially altered the expression of PIN genes,
and particularly increased PIN2 expression in the root (Peer
et al., 2004). Consistent with this, elevated auxin transport
in mutants lacking flavonoids, endogenous auxin transport
inhibitors, resulted in increased IAA levels in the root
which, in turn, enhanced PIN expression and altered PIN
protein localization (Peer et al., 2004).
A number of auxin transport inhibitors have been used as
pharmacological agents to probe the subcellular trafficking
of PIN proteins. The competitive inhibitor triiodobenzoic
acid (TIBA) and the non-competitive inhibitor pyrenoyl
benzoic acid (PBA) both disrupt PIN vesicle trafficking
processes by interfering with actin stability (Dhonukshe
et al., 2008), and TIBA can inhibit restoration of PIN1 to
the plasma membrane when included in washout solutions
following BFA treatment (Geldner et al., 2001). NPA can
function in a similar fashion if used in even higher
concentrations (Geldner et al., 2001; Petrasek et al., 2003).
However, in all cases, the concentrations of auxin transport
inhibitors required for inhibition of cellular trafficking
mechanisms are 10–100 times higher than those required to
inhibit auxin transport (Petrasek et al., 2003).
Subcellular trafficking of AUX1
The trafficking of AUX1 exhibits both differences and
similarities when compared to that of PIN proteins.
Although AUX1 exhibits polar localization on the apical
plasma membrane in some cells, in others, AUX1 exhibits
a non-polar membrane distribution and is also accumulated
at the Golgi apparatus and endosomal compartments
(Kleine-Vehn et al., 2006). The connections between these
plasma membrane and intercellular localizations have been
shown to be dependent on actin filaments and the membrane sterols (Kleine-Vehn et al., 2006). Moreover, the
apical localization of AUX1 on the plasma membrane of
protophloem and epidermal cells requires the presence of
AUXIN RESISTANT4 (AXR4), which is found in the
endoplasmic reticulum (ER) (Dharmasiri et al., 2006). In
contrast to AUX1, the localizations of PIN1 and PIN2 did
not appear to be affected by AXR4, thus indicating that the
root agravitropic phenotype observed in the axr4 mutant is
probably caused by an alteration in the AUX1 function.
Although AUX1 trafficking is also BFA-sensitive, it utilizes
a GNOM-independent mechanism (Kleine-Vehn et al.,
2006; Boutte et al., 2007). However, this is not surprising
considering the likelihood of BFA interaction with Sec7-like
motifs in multiple proteins.
Subcellular trafficking of ABCBs
ABCB19 is localized on the plasma membrane where it
partially overlaps with PIN1 (Blakeslee et al., 2007).
However, the trafficking of ABCB19 does not coincide with
that of PIN1, PIN2, and AUX1. Compared with the more
dynamic processes regulating membrane localization of the
AUX1 and PIN-family proteins, ABCB19 is more stably
situated on the plasma membrane (Titapiwatanakun et al.,
2008). Whereas the dynamic cycling of PIN1 is disrupted by
short-term treatments with actin depolymerising agents like
latrunculin B (Geldner et al., 2001), the localization of
ABCB19 is unaffected by a short-term treatment with
latrunculin B. ABCB19 is not recycled by microtubule- or
SNX1-dependent processes, as ABCB19 subcellular localization is insensitive to short-term treatments with the
microtubule depolymerizing compound oryzalin and is also
insensitive to wortmannin (Titapiwatanakun et al., 2008).
However, treatment with gravacin does interfere with the
trafficking of ABCB19 to the plasma membrane, resulting
in aggregation of some ABCB19 protein in an unidentified
compartment that does not coincide with the endocytic
marker FM4-64 (Rojas-Pierce et al., 2007). Identification of
this compartment should help the elucidation of the
ABCB19 trafficking pathway.
As the trafficking of ABCB19 to the plasma membrane is
not BFA-sensitive, it is not mediated by GNOM-dependent
mechanisms (Titapiwatanakun et al., 2008). However, not
surpisingly for a P-glycoprotein, ABCB19 appears to be
trafficked by GNOM-LIKE1 (GNL1), a BFA-insensitive
ARF-GEF in the GNOM family that mediates vesicular
ER-Golgi trafficking (Richter et al., 2007; Teh and Moore,
2007). In Arabidopsis, mutations in GNL1 exhibit a reduction in the abundance and plasma membrane localization of ABCB19, but not of PIN1 and PIN2. The gnl1
mutants also exhibited a decrease in NPA-sensitive auxin
transport.
On the other hand, ABCB1 does aggregate in intracellular bodies with PIN2 after BFA treatment, suggesting that
it is less stable and more readily endocytosed than ABCB19
(Blakeslee et al., 2007; Titapiwatanakun et al., 2008). This is
consistent with the synergistic phenotypic effects seen with
loss of ABCB1 function in abcb19 mutants and suggests
that ABCB1 may be a more dynamic regulator of ABCB19
function. This is even more likely, as ABCB1 exhibits
stronger interactions with both FKBP42/ TWD1 and the
Post-transcriptional regulation of auxin transport proteins | 1101
auxin transport inhibitor NPA (Murphy et al., 2002; Geisler
et al., 2003; Bouchard et al., 2006; Bailly et al., 2008).
Recently, the involvement of small GTPases, such as the
Rab proteins, in the trafficking of human ABCBs has been
reported in a HeLa cells study (Fu et al., 2007). The
intracellular localization of human ABCB1 became conspicuous when a dominant negative form of Rab5 and GFPtagged HsABCB1 were co-expressed in HeLa cells (Fu
et al., 2007). It will be interesting to investigate further
whether the small GTPase family of proteins plays a role in
regulating the vesicular trafficking of the ABCBs in plants.
Regulation of auxin transporters by protein
phosphorylation
Protein phosphorylation and dephosphorylation are posttranslational modifications that are commonly used to
regulate activity and/or function of a particular protein.
The serine-threonine kinase PINOID has been shown to
promote the polar localization of PIN1 and the pinoid mutant
exhibits a similar phenotype to that of pin1 (Christensen
et al., 2000). The apical and basal membrane localization of
PIN1 were also shown to be manipulated by the overexpression and inactivation of PID (Friml et al., 2004).
Therefore, PID appears to function as a binary switch that
can reverse the direction of auxin movement. The activation
of PID, in turn, requires phosphorylation of the protein by
a 3-phosphoinositide-dependent protein kinase 1 (PDK1)
(Zegzouti et al., 2006). PIN proteins also appear to be
regulated by dephosphorylation catalysed by the trimeric
serine-threonine protein phosphatase 2A (PP2A) (Michniewicz et al., 2007). A regulatory subunit of PP2A is encoded
by the ROOT CURLING IN NPA1 (RCN1) gene. The rcn1
mutant exhibits a decrease in PP2A activity, defects in root
and hypocotyl elongation, and altered apical hook formation
(Garbers et al., 1996). This suggests that PP2A may play a
role in NPA-sensitive root acropetal auxin transport (DeLong
et al., 2002).
The linker region between the two repeated nucleotide
binding domains of ABCB proteins is a prominent target
for phosphorylation in yeast and humans (Ambudkar et al.,
1999). In Arabidopsis, phosphoproteomics of plasma membrane proteins revealed three possible sites in ABCB proteins that can be phosphorylated by related protein kinases
(Nuhse et al., 2004). One potential candidate is PHOTOTROPIN1 (PHOT1), a plasma membrane serine-threonine
protein kinase that functions in multiple blue-light responses (Inoue et al., 2008). The PHOT1 protein exhibits
an intracellular localization upon blue-light illumination
(Briggs and Christie, 2002; Wan et al., 2008) that is concurrent with the delocalization of PIN1 in vascular tissues
of photoresponding hypocotyls (Blakeslee et al., 2004).
However, to date there is no evidence of direct interactions
between PHOT1 and PIN1. However, ABCB19 may
regulate this interaction, as ABCB19 stabilizes PIN1 on the
plasma membrane (Titapiwatanakun et al., 2008), and
abcb19 mutants exhibit more rapid phototropic responses
in hypocotyls (Noh et al., 2003; Lin and Wang, 2005;
Nagashima et al., 2008). Another potential target of
PHOT1 is PIN3, which is thought to contribute to lateral
translocation of auxin (Friml et al., 2002b). PIN3 localization in hypocotyl epidermal cells, whose elongation is
differentially regulated in tropic bending, suggests that
PHOT1 functions in inactivating localized efflux of auxin
mediated by PIN3 in cells below the site of illumination.
However, blue light responses mediated by PHOT1 do not
appear to regulate membrane localization of ABCB19 or
PIN3, as, after the blue-light treatment, no alterations in the
subcellular localization of ABCB19 and PIN3 are observed,
whether native, epitope-tagged, or YFP/GFP translational
fusion proteins are monitored (not shown).
Regulation of auxin transporters by
ubiquitin-mediated proteolysis
The steady-state levels of many proteins that are implicated
in auxin transport are actively regulated by ubiquitinmediated protein degradation. The post-transcriptional
stability of PIN proteins has been shown to require the
presence of MODULATOR OF PIN (MOP) proteins
(Malenica et al., 2007). The Arabidopsis mop2 and mop3
mutants were identified in a genetic screen in the eir1-1
mutant background for mutations that affect auxin responses and/or distribution. In addition to a root agravitropic phenotype, the mop2 and mop3 mutants also
exhibited auxin-related phenotypes similar to those seen in
pin mutants. Further characterization of these mutants
showed a decrease in the abundance of PIN proteins, such
as PIN1, PIN2, and PIN3, suggesting that MOP played an
essential role in regulating the steady-state level of PIN
proteins.
Cumulative evidence suggests that PIN2 is selectively
regulated by ubiquitination (Abas et al., 2006). Gravistimulation of roots results in a greater abundance of plasma
membrane-localized PIN2 in epidermal cells on the underside of the root. On the upper side of the root, PIN2 was
found in an intracellular compartment as well as on the
plasma membrane. This internalization of PIN2 appears to
be controlled by proteasomic activity, as the internalization
and degradation of PIN2 was prevented by pre-treatment
with the proteasome inhibitor MG132 (Abas et al., 2006).
Regulation of PIN1 by ubiquitin-mediated turnover is
suggested by the presence of a lower molecular weight band
(;50 kDa) typically observed in immunoblot analyses of
PIN1 that is prevented by treatment with inhibitor cocktails
containing MG132 (Titapiwatanakun et al., 2008). The
presence of PIN1 proteolytic products may also explain
differences observed between immunolocalizations of PIN
proteins, which all utilize antisera generated against the central ‘soluble’ loop of the proteins, and some PIN-fluorescent
protein fusions. Proteolytic turnover may also play a role in
the stabilization of PIN1 associated with ABCB19-containing
membrane subdomains, as PIN1 is more resistant to degradation in those fractions.
1102 | Titapiwatanakun and Murphy
Regulation of auxin transporters by
flavonoids
The effect of NPA can be mimicked by the activity of
endogenous flavonoids, which can inhibit polar auxin transport and modify local auxin concentrations (Murphy et al.,
2000). In addition, flavonol compounds, such as quercetin
and kaempferol, can be displaced by NPA from the microsomal extract of Arabidopsis plants. Endogenous flavonols
have also been shown to regulate auxin transport negatively
and alter gene expressions and subcellular localization of
PIN1, PIN2, and PIN4 (Peer et al., 2004; Peer and Murphy,
2007). To date, no evidence of flavonoid regulation of
AUX1 has been reported.
The relationship between flavonoids and ABCB proteins
has been demonstrated, as the addition of flavonols inhibits
ABCB-mediated auxin transport in heterologous expression
systems (Peer and Murphy, 2007). In abcb4 and abcb19
mutants, aggregations of quercetin accumulates in the same
region where ABCB4 and ABCB19 would be localized
(Terasaka et al., 2005; Titapiwatanakun et al., 2008). ABCB
proteins, particularly ABCB4, are likely to be the principal
targets of flavonoid regulation of auxin transport at the
plasma membrane. Although the flavonoid-deficient transparent testa4 tt4 mutant displayed a slower gravitropic response compared to the wild type, the gravitropic response
of abcb4 tt4 double mutant resembled that of abcb4, indicating that abcb4 was epistatic to tt4 (Lewis et al., 2007).
Regulation of auxin transporters by
membrane composition
The mutant lacking STEROL METHYTRANSFERASE1
(SMT1) exhibits aberrant cell polarity, auxin distribution,
and embryo development (Willemsen et al., 2003). SMT1 is
required for the first step of sterol biosynthesis and for the
correct membrane localization of PIN1 and PIN3, but not
for localization of AUX1. The loss-of-function cpi1-1 mutant, which is deficient in the cyclopropylsterol isomerase1
that catalyses a step following SMT1 in the sterol biosynthesis pathway, was also recently shown to exhibit
defects in PIN2 polarity (Men et al., 2008). In normal
cytokinetic epidermal cells, PIN2 is detected in both apical
and basal membranes, whereas, in the post-cytokinetic
epidermal cells, PIN2 is localized only at the apical membrane. However, in the cpi1-1 mutant, where PIN2 endocytosis is altered, the subcellular localization of PIN2
following cytokinesis is associated with the cell-plate-like
structure. Sterols internalized from the plasma membrane
colocalize with the PIN2 recycling endosome and are both
BFA-sensitive and actin-dependent, suggesting a link between endocytic sterol trafficking and PIN polarity (Grebe
et al., 2003). However, not all phenoytpes resulting from
altered sterol trafficking are derived from altered auxin
transport. A mutation in a gene encoding an Arabidopsis
sterol carrier protein-2 was recently shown to regulate
normal seed and seedling metabolism (Zheng et al., 2008).
A defect in myo-inositol-1-phosphate synthase (MIPS)
results in an altered embryogenesis, venation patterning,
and root growth. MIPS plays a crucial role in inositol
biosynthesis. Thus, it has been suggested that the phosphoinositide levels of the plasma membrane can directly
impact polar auxin transport. Mutants lacking MIPS
showed a slower rate of endocytosis and a higher degree
of wortmannin sensitivity in PIN2-positive endosomes,
thereby providing evidence for a contribution of MIPS to
the regulation of vesicular trafficking in plants.
Specialized microdomains that are characteristically
enriched in sterol lipids and remain soluble in high concentrations of detergents have been reported (Mongrand
et al., 2004; Borner et al., 2005). These microdomains
have also been shown to function in the association of
membrane proteins and in endocytosis (Morel et al.,
2006). Sterols and sphingolipids constitute a principal
factor in the formation of plasma membrane microdomains (Heese-Peck et al., 2002; Jaillais and Gaude,
2008). Although the evidence that supports the existence
of plasma membrane microdomains in plants is relatively
limited, the lipid composition of the detergent-resistant
membrane (DRM) fraction from Arabidopsis membranes
is enriched in sterols, as well as glucosylceramide. A link
between sterol composition and the endocytotic pathway
has also been reported (Sharma et al., 2002). More
recently, functional sphingolipid synthesis and trafficking
has been shown to be required for normal plant development and growth (Chen et al., 2008), suggesting that
sphingolipid content may also be an important factor in
regulating membrane domains.
DRM fractions derived from plant cell cultures were
shown to contain ABCB and PIN proteins (Mongrand
et al., 2004; Borner et al., 2005; Morel et al., 2006). Interestingly, the localization of PIN1 in DRMs requires
intact ABCB19, as PIN1 was undetectable in DRM fractions derived from abcb19 seedlings and mature plants (Fig.
3; Titapiwatanakun et al., 2008). The abcb19 mutant also
exhibits an altered rate of endocytosis compared to the wild
type. As the DRM-associated proteins in mammals are
thought to be sorted and internalized via a caveolin-dependent pathway (Sharma et al., 2002), the presence of
ABCB19 in DRMs suggests that it may not only stabilize
membrane microdomains, but also, indirectly, regulate
endocytotic processes. Consistent with this interpretation,
some BFA-induced aggregates containing PIN1 remain
intact in abcb19 after BFA is washed out, suggesting that
ABCB19 also has an important function in reconstitution of
the endosomes to the plasma membrane (Titapiwatanakun
et al., 2008).
The importance of sterols in defining the proper
membrane environment may explain the difficulty encountered in attempting biochemically to demonstrate PIN1
auxin efflux activity by heterologous expression in multiple
host organisms (Petrasek et al., 2006; Blakeslee et al.,
2007). However, Schizosaccharomyces pombe was recently
found to be suitable for the expression of PIN1 as a fully
active transporter (Titapiwatanakun et al., 2008). This
Post-transcriptional regulation of auxin transport proteins | 1103
Acknowledgements
We thank Dr Wendy Ann Peer for a careful reading of the
manuscript. This work is supported by the US Department
of Energy to ASM.
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