1

High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter

in Arabidopsis thaliana

Koji Kasai‡, Junpei Takano§, Kyoko Miwa‡,1, Atsushi Toyoda‡,2, Toru Fujiwara‡, ¶, !,3

From the ‡Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan,

the §Research Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan,

the ¶Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657,

Japan.

the Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology

Agency, Saitama 332-0012, Japan.

Running Head: Ubiquitination of BOR1 for vacuolar sorting 1Present address: Creative Research Institution, Hokkaido University Kita-ku, Sapporo, 001-0021,

Japan 2Present address: Food Technology Department, Nagano Prefecture General Industrial Technology

Center, 205-1 Aza-Nishibanba, Oaza-Kurita, Nagano-shi, Nagano 380-0921 Japan. 3Address correspondence to: Toru Fujiwara, Department of Applied Biological Chemistry, Graduate

School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657 1-1-1 Yayoi,

Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: 81-3-5841-5104; Fax: 81-3-5841-8032; E-mail:

atorufu@mail.ecc.u-tokyo.ac.jp

Boron (B) homeostasis is important for plants,

as B is essential but is toxic in excess. Under

high-B conditions, the Arabidopsis thaliana

borate transporter BOR1 is trafficked from

the plasma membrane (PM) to the vacuole via

the endocytic pathway for degradation to

avoid excess B transport. Here, we show that

B-induced ubiquitination is required for

vacuolar sorting of BOR1. We found that a

substitution of lysine 590 with alanine

(K590A) in BOR1 blocked degradation.

BOR1 was mono- or diubiquitinated within

several minutes after applying a high

concentration of B, whereas the K590A

mutant was not. The K590A mutation

abolished vacuolar transport of BOR1 but

did not apparently affect polar localization to

the inner PM domains. Furthermore,

brefeldin-A and wortmannin treatment

suggested that K590 is required for BOR1

translocation from an early endosomal

compartment to multivesicular bodies

(MVBs). Our results show that the B-induced

ubiquitination of BOR1 is not required for

endocytosis from the PM but is crucial for

sorting of internalized BOR1 to MVBs for

subsequent degradation in vacuoles.

Although boron (B) is an essential

micronutrient for plants, excess B is toxic. The

homeostasis of B is important for plants. In fact,

http://www.jbc.org/cgi/doi/10.1074/jbc.M110.184929The latest version is at JBC Papers in Press. Published on December 9, 2010 as Manuscript M110.184929

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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both B deficiency and toxicity have negative

impacts on agricultural production (1, 2). The

only established function of B in vascular plants

is cross-linking of the pectic polysaccharide

rhamnogalacturonan II (RG-II). This

cross-linking mediated by borate is important to

maintain cell-wall architecture and is necessary

for normal growth and development (3, 4). In

contrast, the molecular mechanism of B toxicity

is still unclear despite several efforts applying

biochemical, genetic, and molecular approaches

(5-9).

B is taken up by plant roots mainly in the

form of boric acid. Arabidopsis thaliana

possesses two distinct types of B-transporting

proteins, BOR1 (10) and NIP5;1 (11), for

effective B uptake into roots and transport to

shoots under B-limiting conditions (12). BOR1

is an efflux-type borate transporter with

similarities to animal bicarbonate transporters

(13). In roots, BOR1 is polar localized to the

inner (stele-facing) plasma membrane (PM)

domain of various cells (14) and functions in

inward transport of B toward the stele under

low-B conditions (10). NIP5;1 is a member of

the major intrinsic protein (MIP) family and

functions as a boric acid channel for efficient B

uptake into the root under B-limiting conditions

(11). NIP5;1 is polar localized to the outer

(soil-facing) PM domain of root epidermal and

root cap cells (14) and increases the permeability

of the outermost PM domain to boric acid for

efficient B uptake.

These transporters have been successfully

used to generate B-stress-tolerant plants. BOR1

overexpression in A. thaliana results in

enhanced B translocation from the root to shoot

and improved shoot growth and seed yields

under B-limiting conditions (15). Enhancing

NIP5;1 expression by activation tag improves

root growth under B-limiting conditions (16).

Overexpression of BOR4, a BOR1 paralog,

reduces B concentration in both roots and shoots

and confers tolerance to toxic levels of B (17).

Accumulations of NIP5;1 and BOR1 are

regulated by B availability via different

regulatory mechanisms. NIP5;1 is regulated at

the level of mRNA accumulation in response to

B concentration in the media (11), whereas

BOR1 accumulation is regulated through protein

degradation. In the presence of high B

concentrations, BOR1 is rapidly removed from

the PM and transferred to the lytic vacuole

through the endocytic pathway for prompt

degradation (18). The endocytic degradation

system is assumed to be beneficial for plants to

quickly shut down B transport and avoid excess

accumulation of B.

Endocytic membrane trafficking is a key

mechanism to control protein amount in the PM.

During endocytic degradation, the target protein

is internalized from the PM to the early

endosomal compartments by endocytosis, sorted

into late endosomes (LEs)/multivesicular bodies

(MVBs), and transferred into

vacuoles/lysosomes for degradation (19).

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Although the details of this mechanism have

been intensively studied in yeast and mammals

(19-22), much less knowledge has accumulated

in plants. A recent report demonstrated that the

A. thaliana brassinosteroid insensitive1 (BRI1)

receptor and BOR1 are sorted into LEs/MVBs

for vacuolar degradation through the

trans-Golgi/early endosome (TGN/EE) network

(23). Ubiquitination is a key signal for the

homeostatic regulation of proteins. Two distinct

forms of ubiquitination are known:

polyubiquitination is used in the proteosomal

protein degradation system, and mono- and

multi-monoubiquination are required for

endocytic internalization of transmembrane

proteins for lysosomal/vacuolar turnover of

membrane proteins in yeast and mammals (19).

In addition, monoubiquitination has also been

reported as a sorting signal of the membrane

proteins to MVBs in mammals (24). This

ubiquitination is required for binding to the

endosomal protein sorting complex (ESCRT). In

A thaliana, the ESCRT-related proteins

CHMP1A and CHMP1B are required for MVB

sorting of the three auxin carriers PIN1, PIN2,

and AUX1 (25). PIN2 turnover, which is related

to root gravitropism, is regulated by endocytic

membrane trafficking and proteasome activity

(26). This study showed that PIN2 is modified

by ubiquitin, and that ubiquitination depends on

proteasome activity; however, whether

polyubiquitination or multi-monoubiquitination

is required remains unclear. In addition, no

direct evidence exists that ubiquitination is

involved in PIN2 degradation (26).

Here, we identified an ubiquitination site

in BOR1, which is required for B-induced

vacuolar sorting of BOR1. We previously

reported that BOR4, unlike BOR1, is not

degraded by high-B (+B) supplementation (17).

Thus, in this study, we searched amino acid

residues involved in B-induced degradation of

BOR1 by differences between the BOR1 and

BOR4 sequences, and found that lysine 590

(K590) was critical for the degradation.

Supplementation with high-B induced mono- or

diubiquitination of GFP-tagged BOR1

(BOR1-GFP), and a K590 mutation completely

inhibited ubiquitination. Furthermore, the K590

mutation did not inhibit the endocytosis step

from the PM but prevented MVB sorting of

BOR1 for degradation in the vacuole.

MATERIALS AND METHODS

Plasmid construction and plant

transformation–Binary plasmids for plant

transformation to express GFP-fused BOR

mutants under control of the BOR1 promoter

were generated using Gateway technology

(Invitrogen, Carlsbad, CA, USA). Construction

of BOR1–BOR4 chimeras and site-directed

mutagenesis were conducted using the in vitro

overlap–extension PCR method (27).

BOR1-GFP (10) or BOR4-GFP (17) constructs

were used as templates, and the primers used are

listed in Supplemental Table I. The PCR

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fragments of GFP-tagged genes were subcloned

into pENTR/D-TOPO (Invitrogen) according to

the manufacturer’s instructions and sequenced to

confirm that that no PCR error occurred. The

subcloned GFP-tagged genes were mobilized

into pAT100 (14), a destination binary vector

carrying the BOR1 promoter, with LR clonase

(Invitrogen), according to the manufacturer’s

instructions.

The resulting binary plasmids were

introduced into Agrobacterium tumefaciens

strain GV3101:pMP90 (28). A. thaliana

bor1-3/2-1 plants, a BOR1/BOR2 double null

mutant, were transformed using the floral dip

method (29).

To generate a T-DNA insertion mutant for

both BOR1 and BOR2, T2 seeds of bor1-3

(SALK_37312) and bor2-1 (SALK_56473)

were obtained from the Salk Institute (30)

(http://signal.salk.edu/cgi-bin/tdnaexpress).

bor1-3 and bor2-1 are in the Col-0 background

and carry T-DNAs in their exons. The plants

were crossed and those homozygous for both

T-DNAs were selected by PCR. The primers

used for PCR were for bor1-3, (BOR1 forward)

5'-GCATACCAAGAGCTTAGCAACT-3',

(BOR1 reverse)

5'-TGGAGTCGAACTTGAACTTGTC-3' and

the SALK left border (LBb1),

5'-GATGGCCCACTACGTGAACCAT-3', for

bor2-1, (BOR2 forward)

5'-CATGCAACAAGCCATCAAAG-3', (BOR2

reverse) 5'-AAATCCAAGAAGAAGCAGAT-3'

and the SALK left border (LBb1).

Plant growth conditions–MGRL medium (31)

was prepared with slight modifications

according to Takano et al (18) and used with

various concentration of boric acid as the B

source. MGRL solid medium was made with

1.5% gellangum (Wako Pure Chemical, Inc.

Osaka, Japan) and 2% sucrose in a sterile square

Petri dish (140 ! 100 mm; Eiken, Tokyo, Japan).

A thaliana seeds were surface-sterilized by

soaking them in 99.5% ethanol for 2 min and

allowed to air-dry for a few hours; then they

were sown onto MGRL solid medium. After a

2-day cold acclimation at 4°C, the plates were

placed vertically, and the plants were grown at

22°C under a 16 h light:8 h dark cycle.

For microsome isolation, plants were grown

on vertically placed MGRL solid medium

containing 30 "M B for 16 days and were

hydroponically grown with MGRL liquid

medium containing 30 "M B for 4 days and then

with the medium containing 3 "M B (–B) for 4

days under a 10 h light:14 h dark cycle, followed

by treatment with the medium containing 100

"M B (+B, an optimal condition). The method

for hydroponic culture was as follows.

Twenty-five plants were placed between two

formed polyethylene sheets (15 ! 100 ! 4 mm),

and four sets of the sheets were floated on 400

ml of MGRL liquid medium in a light-shielded

plastic container. The liquid medium was

continuously aerated by an air pump and was

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changed every 4 days.

Confocal fluorescent imaging–Confocal imaging

was performed as described previously with

slight modifications (18). The transgenic plants

were grown on MGRL solid medium

supplemented with 3 "M B (–B) for 4 days. The

roots were cut and incubated with MGRL liquid

medium containing boric acid, inhibitors, or

fluorescent dye at room temperature, as

described in the figure legends. A stock solution

of cycloheximide (CHX) or FM4-64 (Molecular

Probes, Carlsbad, CA, USA) was prepared with

water at 25 mM or 10 mM respectively, and

BFA (Sigma, St. Louis, MO, USA) was

prepared with DMSO at 50 mM.

Confocal imaging was performed using an

LSM510 (Carl Zeiss, Oberkochen, Germany) or

an FV1000 laser scanning confocal microscope

(Olympus, Tokyo, Japan). The excitation

wavelength was 488 nm, and emitted

fluorescence was detected with a 505–530-nm

band-pass filter for GFP or a 650-nm long-pass

filter for FM4-64. Signal intensity was

monitored to avoid pixel saturation.

At least two independent transgenic lines

(T2 or T3 plants) from each construct were used

to confirm the reproducibility of the results.

Preparation of microsomal fractions from

Arabidopsis tissues–Microsomal proteins were

prepared as described previously with some

modifications (18). The bor1-3/2-1 and

transgenic plants (T3 homo-lines) were grown

hydroponically, as described above. The roots

and shoots were harvested after treatment with

100 "M B (+B) MGRL liquid medium.

Approximately 1 g of tissues was homogenized

in 5 ml of ice-cold homogenization buffer (250

mM Tris-Cl [pH 7.8], 25 mM

ethylenediaminetetraacetic acid [EDTA], 290

mM sucrose, 75 mM 2-mercaptoethanol, 10 mM

N-etylmaleimide, 1 mM phenylmethylsulfonyl

fluoride [PMSF], and one tablet/50 ml of

protease inhibitor cocktail [complete,

EDTA-free; Roche, Indianapolis, IN, USA])

using a POLYTRON PT-2100 homogenizer

(Kinematica Inc., Bohemia, NY, USA) at 26,000

rpm for 5 s. This homogenization was repeated

four times. The homogenates were centrifuged at

8,000 ! g for 5 min at 4°C, and the supernatant

was centrifuged at 100,000 ! g for 10 min at

4°C. The microsome pellets were resuspended in

200 "l of ice-cold storage buffer (50 mM Tris-Cl

[pH 8.0], 150 mM NaCl, 10 mM

N-etylmaleimide, 1 mM PMSF, and one

tablet/50 ml of protease inhibitor cocktail

[complete, EDTA-free; Roche]) using a pestle.

The protein concentration was determined using

the Bradford method (Quick Start Protein Assay

Kit I; Bio-Rad, Hercules, CA, USA). The

samples were frozen in liquid nitrogen and

stored at –80°C.

Immunoblot analysis–Microsomes containing 20

"g of protein were dissolved in 2! solubilization

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buffer (50 mM Tris-Cl [pH 7.5], 50 mM

dithiothreitol [DTT], 5% glycerol, 5% sodium

dodecyl sulfate [SDS], 5 mM EDTA, 0.03%

bromophenol blue, one tablet/10 ml of protease

inhibitor cocktail [complete mini, EDTA-free;

Roche]), incubated at 65°C for 5 min, and then

separated by SDS-polyacrylamide gel

electrophoresis (PAGE) using a modified

Laemmli system (32) with 8% acrylamide and

0.064% N,N'-methylene-bis-acrylamide. Gels

were blotted onto polyvinylidene fluoride

(PVDF) membranes (BioCraft, Tokyo, Japan) by

semidry electroblotting. The anti-GFP mouse

monoclonal antibody GF200 (Nakalai Tesque,

Kyoto, Japan) was used at 2.2 "g/ml in 2% ECL

advance blocking reagent (GE Healthcare,

Piscataway, NJ, USA). Immune complexes were

detected with horseradish peroxidase-conjugated

goat antibody to mouse IgG and ECL advance

reagents (GE Healthcare), and the

chemiluminescence was detected with an

LAS-3000 imaging system (Fujifilm, Tokyo,

Japan).

Immunoprecipitation and detection of

ubiquitination–Microsomal protein fractions (50

"g) were lysed in 300 "l of lysis buffer (20 mM

HEPES-KOH [pH 7.5], 150 mM NaCl, 1 mM

EDTA, 1% Tween 20, 0.5% deoxicholate, 0.1%

SDS, 10 mM N-ethylmaleimide, 1 mM PMSF,

and one tablet/10 ml of protease inhibitor

cocktail [Complete mini, EDTA-free; Roche]),

and incubated with 20 "l of agarose conjugated

anti-GFP monoclonal antibody (50% gel slurry;

MBL, Nagoya, Japan) for 1.5 h at 4°C with

gentle shaking. After the incubation, the beads

were washed three times with lysis buffer, and

the bound proteins were analyzed by

immunoblotting using anti-GFP antibody or

anti-ubiquitin (Ub) antibody. Anti-Ub

monoclonal antibody (P4D1; Santa Cruz

Biotechnology, Santa Cruz, CA, USA) was used

at 0.5 "g/ml in 2% ECL advance blocking

reagent.

RESULTS

The Amino Acid Sequence Involved in

Boron-Induced Degradation of BOR1–Our

previous studies demonstrated that BOR1 and

BOR4 are distinct in terms of B-induced

endocytic degradation and polar localization (14,

17); BOR1 and BOR4 localize to the inner and

outer PM domains of various root cells,

respectively, and BOR1 is degraded by +B

supplementation (Fig. 2), but BOR4 is not.

Therefore, we searched amino acid residues

involved in B-induced degradation by means of

differences between BOR1 and BOR4

sequences.

To identify the BOR1 region responsible

for B-induced degradation, we constructed two

BOR1 and BOR4 chimeras. The C-terminal

region of BOR1 was replaced with the

corresponding region of BOR4 from L468 or

E641 and called chimera 1 and chimera 2,

respectively (Fig 1). The BOR1, BOR4, and

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chimeras were C-terminally fused to GFP and

placed under the control of the BOR1 promoter

were generated. To exclude the possibility that

an endogenous BOR1 or BOR2, the closest

homolog of BOR1, disturb the mutational effects

of introduced BORs through protein–protein

interactions, the constructs were introduced into

bor1-3/2-1, a double BOR1 and BOR2 mutant

without BOR1 and BOR2 accumulation. The

growth defect in the bor1-3/2-1 mutant under

B-limiting conditions was restored almost fully

by expressing BOR1-GFP and partially restored

by BOR4-GFP, chimera 1-GFP, and chimera

2-GFP (Fig. S1), indicating that these expressed

proteins possess B-transporting activity. As

shown in Fig. 2, BOR1-GFP was localized to the

PM of the root cap and epidermal cells in the

root tip with inward polarity and degraded upon

treatment with +B for 2 h. In contrast, chimera

1-GFP and BOR4-GFP did not show inward

polarity and did not disappear with +B.

Although a part of the chimera 1-GFP was

observed in the cytosol or endomembrane, most

likely endoplasmic reticulum (ER), PM

localization was confirmed by co-localization

with a specific membrane dye FM4-64 (33) (Fig.

S4). Chimera 2-GFP showed no or a weaker

polarity but was degraded by +B

supplementation. These results suggest that the

BOR1 sequence from L469 to E641 and F642 to

the last amino acid residue (N704) contains the

motif(s) for B-induced degradation and polar

localization, respectively.

Lysine 590 Is Crucial for Boron-Induced

Degradation of BOR1–We focused on potential

ubiquitination sites to identify an amino acid

residue required for boron-induced endocytic

degradation of BOR1. Ubiquitin conjugation to

the lysine residue in yeast is involved in

endocytosis and degradation of PM proteins

such as receptors, channels, and transporters (22,

34). Therefore, we searched for a lysine residue

in the region from L469 to E641 of BOR1 and

found only one lysine residue, K590, which is

not conserved in BOR4 (Fig. 1B). We

introduced a mutation to change K590 to A

(K590A). The GFP-tagged K590A mutant

(BOR1K590A-GFP) was expressed under the

control of BOR1 promoter. Several independent

transgenic plants were generated, and the

transgenic plant lines displayed almost full

restoration of the bor1-3/2-1 growth defect

under a B-limiting condition (Fig. S1). The

BOR1K590A-GFP showed inward polarity similar

to the wild-type BOR1-GFP, however,

B-induced degradation was not observed (Fig.

3A). GFP fluorescence was seen in the PM 4

days after treatment with +B (100 "M). This

result indicates that K590 is involved in BOR1

degradation. For further confirmation,

microsomes isolated from shoots and roots of

these transgenic plants after treatment with +B

were subjected to immunoblot analysis using an

anti-GFP antibody (Fig. 3C). A single band

corresponding to the expected size of full-length

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BOR1-GFP or BOR1K590A-GFP (approximately

106 kDa) was detected in all root samples

indicating that the GFP fluorescence seen in Fig.

3A was derived from the full-length BOR1-GFP

protein. B-induced degradation of BOR1-GFP

was observed in the root samples but

degradation was not detected for

BOR1K590A-GFP, consistent with the results of

GFP imaging. Degradation was not observed in

either BOR1-GFP or BOR1K590A-GFP in the

shoot during this short period of time (30–120

min). In the shoot, another band was observed at

a higher weight than 250 kDa, which was

assumed to be a trimer or tetramer.

We also tested the effects of other lysine

mutations in the BOR1 C-terminal region (see

Fig. 1B), including K649A, K689A, and a

K649A/K689A double mutant. All three mutant

proteins, BOR1K649A-GFP, BOR1K689A-GFP, and

BOR1K649A,K689A-GFP, fully complemented

hypersensitivity to B deficiency of the

bor1-3/2-1 mutant (Fig. S1) and exhibited

normal polarity and B-induced degradation (Fig.

S2). These results further confirmed the

importance of K590, but not of other nearby

lysine residues.

We then tested whether K590 is sufficient

for B-induced degradation of BORs by

introducing an N590K or N590K/P591G double

mutation in chimera 1-GFP and N596K or

N596K/P597G in BOR4-GFP. These changes

represent the introduction of BOR1-type amino

acid residues at these specific positions in the

BOR4-type sequences. These constructs

partially rescued the hypersensitivity to B

deficiency of the bor1-3/2-1 mutant, confirming

B-transport activity of the BORs-GFP

derivatives (Fig. S1). These amino acid

substitutions did not induce B-dependent

degradation in both chimera 1-GFP and

BOR4-GFP (Fig. S3). The chimera 1 derivatives,

chimera 1N590K-GFP and chimera 1N590K,

P591G-GFP, as well as chimera 1-GFP, were

localized in the PM and partly in the

endomembrane, most likely the ER. The PM

localization of these proteins was further

confirmed by co-localization with FM4-64 (Fig.

S4). These results indicate that K590 is

necessary but not sufficient for B-induced

degradation of BORs.

BOR1 Is Ubiquitinated Under +B Conditions,

and Lys 590 Is the Principal Ubiquitination

Site–We assumed that BOR1 ubiquitination at

K590 was involved in B-induced degradation.

Therefore, we attempted to detect ubiquitin

conjugated to BOR1-GFP and compared that to

BOR1K590A-GFP. BOR1-GFP, BOR1K590A-GFP,

and BOR1K649A,K689A-GFP were

immunoprecipitated using the anti-GFP antibody

from root extracts of transgenic plants after a

time-course treatment with +B, and the

ubiquitination was detected by immunoblotting

using an anti-Ub antibody (Fig. 4). Two specific

ubiquitinated protein bands were observed from

BOR1-GFP and BOR1K649A,K689A-GFP within 30

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min after treatment with +B. The lower and

upper bands corresponded to the size of

BOR1-GFP conjugates with one and two

ubiquitin molecules, respectively. Apparently no

ubiquitinated protein was observed for

BOR1K590A-GFP. In addition, immunoblotting

using anti-GFP detected a relatively small

amount of diubiquitinated BOR1-GFP compared

to non-ubiquitinated protein in a long-exposure

image, indicating that the ubiquitinated BOR1

protein is rapidly degraded and does not

accumulate in large amounts within plant cells.

These results suggest that BOR1 is mono- or

diubiquitinated and that K590 is essential for

ubiquitination under a +B condition. Thus, K590

is likely to be the ubiquitination site.

Lysine590 Is Essential for Vacuolar Sorting of

BOR1 But Not for Endocytosis–B-induced

turnover of BOR1 is mediated by endocytosis

and subsequent degradation in the vacuole (18).

To determine which step of the endocytic

degradation of BOR1 requires K590

ubiquitination, we compared the endocytic

trafficking of BOR1-GFP and BORK590A-GFP

using a general endocytosis marker FM4-64 (33)

and membrane traffic inhibitor brefeldin A

(BFA). BFA inhibits the transition of membrane

proteins from early endosomes (EEs) to MVBs

and to PM but allows endocytosis (35-37)

BFA treatment was performed in the

presence of cycloheximide (CHX) to prevent de

novo protein synthesis. After 30 min of BFA

treatment, both BOR1-GFP and BOR1K590A-GFP

accumulated with endocytosed FM4-64 in the

BFA compartment of root epidermal cells (Fig.

5A). The same volume of dimethyl sulfoxide

(DMSO), the BFA stock solution solvent, was

used as the control, and no effect on traffic was

observed. This result indicated that K590 is not

involved in endocytosis of BOR1 from the PM;

however, it may be involved in subsequent

vacuolar sorting steps (Fig. S6).

To exclude the possibility that K590A

simply enhanced protein synthesis and

overaccumulated BOR1 at the PM, we

monitored GFP accumulation in the root

vacuoles of transgenic plants expressing

BOR1K590A-GFP and compared that with

BOR1-GFP to confirm that the K590A mutation

inhibited BOR1 transport to the lytic vacuole. In

the absence of light, GFP is resistant to vacuolar

proteolysis; thus, dark treatment is an effective

method to visualize vacuolar targeting of

GFP-tagged proteins (14, 36, 38). The roots of

transgenic plants expressing BOR1-GFP and

BOR1K590A-GFP were incubated in low-B (–B)

and +B medium in the dark for 2 h, and GFP

fluorescence in the epidermal cells of the root tip

was monitored (Fig. 5B). After –B treatment,

fluorescence from both BOR1-GFP and

BOR1K590A-GFP was observed in the PM. After

+B treatment, GFP fluorescence in the

BOR1-GFP expressing plant was observed in

the vacuole as previously reported (14), whereas

fluorescence in the BOR1K590A-GFP expressing

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plant was not detected in the vacuole but was

retained in the PM. This observation confirmed

that the K590A mutation prevents sorting of

BOR1 into the vacuole under a +B condition.

We also investigated the effect of

wortmannin (WM) on trafficking of BOR1-GFP

and BOR1K590A-GFP. WM is a specific inhibitor

of phosphatidyl-inositol 3-kinase and induces

homotypic fusions and enlargement of the

prevacuolar compartment (PVC)/LE/MVB in

plants (39-41). In agreement with a previous

study (40), BOR1-GFP was observed in several

round structures after 90 min of 16.5 "M WM

treatment both in +B and –B medium (Fig. 5C,

D) A part of the GFP signal in the round

structures were co-localized with the endocytic

dye FM4-64, indicating that the structures are

enlarged PVC/LE/MVB. In contrast,

BOR1K590A-GFP was rarely observed in

enlarged MVBs. This result suggested that

BOR1K590A-GFP is scarcely able to enter the

MVB pathway.

Taken together, these data suggest that

K590 is not involved in the endocytosis of

BOR1 from the PM but is essential for sorting in

MVBs for subsequent vacuolar degradation (Fig.

S6).

DISCUSSION

We showed that BOR1 is biochemically

modified with mono- or diubiquitin in response

to B availability, and that a K590 residue is

required for both B-induced ubiquitination and

B-induced endocytic degradation. Mutational

analysis showed that a K590A mutant,

BOR1K590A-GFP, was not degraded under +B

conditions unlike BOR1-GFP (Fig. 3), indicating

that K590 is required for B-induced endocytic

degradation. Our most recent work revealed the

other BOR1 residues involved in endocytic

degradation (14): three putative tyrosine-based

sorting signals (YXXØ: Y, tyrosine; X, any

amino acid; Ø, a bulky hydrophobic residue) in

the predicted largest cytosolic loop region. The

tyrosine-based sorting signal is recognized by

the clathrin-adaptor protein (AP) complex for

sorting transmembrane proteins into

clathrin-coated vesicles and is involved in many

post-Golgi trafficking steps including

endocytosis and polar PM trafficking (42).

Mutations in all of the corresponding tyrosines

(Y373A/Y398A/Y405A) of BOR1 prevent

B-induced degradation. Additionally, the

tyrosine mutations inhibit polar localization in

the root tip cells. In contrast, the K590A

mutation inhibits only endocytic degradation and

does not affect polar localization (Fig. 3),

indicating that K590 is specifically required for

BOR1 vacuolar trafficking. We also showed that

introducing an N590K or N590K/P591G

mutation into the chimera 1-GFP and N596K or

N596K/P597G in BOR4-GFP did not promote

B-induced degradation in both chimera 1-GFP

and BOR4-GFP (Fig. S3), indicating that K590

is necessary but not sufficient for endocytic

degradation. Although BOR4 lacks two tyrosine

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residues corresponding to putative

tyrosine-based sorting signals of BOR1, Y373,

and Y405 (Fig. S5), chimera 1-GFP mutants

contain all of the putative tyrosine-based sorting

signals. This suggests the existence of extra

component(s) other than the three tyrosine-based

sorting signals and indicates that K590 functions

in BOR1 endocytic degradation.

Immunoprecipitation and immunoblotting

analyses showed that BOR1 is mono- or

diubiquitinated in a +B-dependent manner and

that K590 is likely a principal ubiquitin-acceptor

site (Fig. 4). Our data are consistent with studies

in yeast showing that several nutrient

transporters are posttranslationally

down-regulated by substrate-dependent mono-

or multi-monoubiquitination (22, 34). Diverse

forms of ubiquitination are involved in the

distinct membrane protein degradation pathways

in yeast and mammalian systems (19);

polyubiquitination mainly regulates proteasomal

degradation, and mono- or

multi-monoubiquitination is required for the

endocytic degradation pathway. In mammals,

K63-linked short-chain ubiquitination and di- or

triubiquitination at the K270 lysine residue

regulates endocytosis of the aquaporin-2 water

channel (43). As a PM localized protein in plants,

PIN2 has been shown to be ubiquitinated (26).

Another report demonstrated that endocytically

internalized PIN2 is degraded mainly in the

vacuole (36). However, for PIN2,

signal-inducing ubiquitination, which is a direct

link between ubiquitination and turnover, the

form of ubiquitination, and the ubiquitination

site are still unknown. Another example is that

downregulation of PIP2, a PM aquaporin, is

regulated via ubiquitination catalyzed by

drought stress-induced Rma1, a homolog of a

RING domain containing E3 ligase in A thaliana

(44). In addition, A thaliana iron regulated

transporter 1 (IRT1), a high affinity iron

transporter, is also posttranslationally regulated

in response to iron availability, and two lysine

residues in the intercellular roop region are

necessary for iron-induced turnover (45).

However, IRT1 ubiquitination has not yet been

reported. Our results show the ubiquitiantion

form, an ubiquitination site, and inducibility by

substrate availability (Fig. 4), thus filling a gap

in the current knowledge regarding

posttranslational turnover of PM proteins in

plants. Our findings suggest that plants are able

to use mono- or diubiquitination as critical

labeling to identify endocytic degradation of PM

proteins.

Our previous report showed that after

treatment with +B for 30–60 min, BOR1-GFP is

translocated into dot-like structures

corresponding to MVBs (18, 23). However,

BOR1K590A-GFP was not observed as dot-like

signals after +B treatment (Fig. 3). Furthermore,

while BOR1-GFP was observed in enlarged

MVBs following WM treatment even under a

–B condition, BOR1K590A-GFP was much less

frequently observed in enlarged MVBs. This

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observation suggests that a pool of BOR1-GFP

is transferred into MVBs even under a –B

condition, and that this MVB sorting is largely

dependent on K590.

In yeast, many PM proteins require mono-

or multi-monoubiquitination for the endocytic

internalization step (22). In contrast,

ubiquitination of epidermal growth factor

receptor (EGFR) in animals is not necessary for

endocytic internalizaion from the PM but is

required for lysosomal sorting (46). Similar to

EGFR, the K590 of BOR1 seems not to be

involved in the endocytosis from the PM but is

essential for transport to the MVB pathway and

then to the vacuole (Fig. 5 and Fig. S6). This

result agrees with the observation that

BOR1K590A-GFP and wild-type BOR1-GFP

showed inward polarity (Fig. 2 and Fig. 3)

because endocytic cycling is required to

maintain polar localization of PM proteins (14,

42). Substrate-induced degradation of number of

yeast nutrient transporters is regulated at the

endocytic internalization step (22). It is

considered to be an efficient way because

unicellular yeast does not require endocytically

generated polarity of nutrient transporters for

efficient nutrient uptake. However, plants might

require continuous endocytic recycling of their

nutrient transporters to maintain polar

localization for directional nutrient transport

under a nutrient-deficient condition. One may

reasonably assume that plants have independent

regulatory mechanisms for endocytosis

(internalization from PM) and vacuolar sorting.

In conclusion, the data presented here show

that B-induced mono- or diubiquitination of

BOR1 at K590 is essential transport step to

MVBs, and the ubiquitination does not control

the rate of endocytosis from the PM, (Fig. S6).

This mechanism is distinct from that of the yeast

system and may be important in enabling polar

localized plant nutrient transporters to achieve a

balance between the polar localization and the

vacuolar degradation systems. BOR1 is a good

model for studying endocytic degradation of

transmembrane proteins in plants because

degradation and ubiquitination can be controlled

by only one amino acid residue. Further studies

may show how plants sense the concentration of

B and regulate ubiquitination in a B-dependent

manner.

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FOOTNOTES

Acknowledgements– We are grateful to Takehiro Kamiya, Kentaro Fuji, and Shimpei Uraguchi for

their helpful discussion and critical reading of the manuscript.

This work was supported, in part, by a Grant-in-Aid for JSPS Fellows (no. 19-7094 to K.K.) from the

Japan Society for the Promotion of Science, a Grant-in-Aid for Scientific Research (to T.F.), and a

Grant-in-Aid for Scientific Research Priority Areas (to T.F.) from the Ministry of Education, Culture,

Sports, Science and Technology, Japan.

The abbreviations used are: B, boron; -B, low-boron; +B, high-boron; PM, plasma membrane; EE,

early endosomes; LE, late endosomes; MVBs, multivesicular bodies; Anti-Ub, Anti-ubiquitin.

FIGURE LEGEND

FIGURE 1 Construction of a BOR1 and BOR4 chimera. A, schematic representation of constructs.

The BOR1 topology model predicted by ConPred II (Arai et al, 2004) is shown at the top. Ten

deduced transmembrane (TM) domains are indicated by black boxes, and the “I” and “o” represent

inside and outside of the cell, respectively. BOR1, BOR4, and two BOR1–BOR4 chimeras, chimera 1

and chimera 2, were tagged with GFP at the C-terminal and inserted under the control of the BOR1

promoter (PBOR1). White, gray, and black segments represent BOR1, BOR4, and GFP sequences,

respectively. The amino acids positions of BOR1 and BOR4 at the chimera junctions are shown. B,

comparison of the C-terminal amino acid sequences of BOR1 (Glu443 to Asn704) and BOR4 (Glu449

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to Glu683). The positions of the two chimera junctions are indicated. The BOR1 unique lysine

residues are denoted by white triangles.

FIGURE 2 Effects of high boron treatment on the subcellular localization of BOR1-GFP,

BOR4-GFP, chimera 1-GFP, and chimera 2-GFP in root cells. A, GFP images from the roots of

transgenic A thaliana expressing BOR1-GFP, BOR4-GFP, chimera 1-GFP, and chimera 2-GFP. Plants

were grown on solid medium with a low boron concentration (–B) and then incubated with –B or

high-B (+B) liquid medium for 2 h. Scale bar represents 50 "m. B, Enlarged views of the epidermal

cells of (A) under the –B condition showing distinct polar localizations. The white arrows indicate the

direction of the polarity. In, Inward; Ou, outward; Bar, 5 "m.

FIGURE 3 Interference with the boron (B)-dependent degradation of BOR1 by a K590A point

mutation. A, GFP images from the roots of transgenic A. thaliana expressing BOR1K590A-GFP. Plants

were grown on solid medium with low B concentration (–B) and then incubated with a high B

concentration (+B) for 2 h (+B2h) or 5 h (+B5h). Plants were also grown on solid medium with a +B

for 4 days (+B4d). Scale bar represents 50 "m. B, enlarged views of the epidermal cells of (A) under a

–B condition. The white arrows indicate the direction of the polarity. In, Inward; Ou, outward; Bar, 5

"m. C, Immunoblot analysis of microsomal fractions from roots and shoots of transgenic plants

expressing BOR1-GFP and BOR1K590A-GFP. The plants were grown on –B medium and then treated

with +B for the indicated time periods. Microsomal proteins were separated by SDS-PAGE and

blotted onto PVDF membranes. The blots were reacted with anti-GFP antibody. Control, microsomes

from bor1-3/2-1. “T” and “M” are the deduced trimer and monomer, respectively. The sizes of the

molecular markers are shown in kDa. CBB-stained membranes are shown as loading controls.

FIGURE 4 Boron (B)-dependent mono- or diubiquitination of BOR1, and involvement of K590

in the ubiquitinations. The microsomal fractions were prepared from the roots of transgenic plants

expressing BOR1-GFP, BOR1K590A-GFP, and BOR1K648A,K689A-GFP, which were grown in low-B (–B)

medium and then treated with a high-B (+B) concentration for the indicated times. The GFP-tagged

proteins were immunoprecipitated with anti-GFP antibody, and ubiquitination was assessed by

immunoblotting with anti-Ubi antibody. “m” and “d” represent the presumed band sizes for the

monoubiquitination and diubiquitination, respectively. Arrowhead indicates the presumed

diubiquitination band detected during a long image exposure of anti-GFP.

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FIGURE 5 Effects of the K590A mutation on the intracellular membrane trafficking of BOR1. A,

Intracellular distribution of BOR1-GFP and BOR1K590A-GFP after treatment with brefeldin A (BFA).

The roots from transgenic plants grown on the low-boron (–B) medium were incubated with 50 "M

cycloheximide (CHX) for 30 min and then with 25 "M FM4-64 for 2 min followed by treatment with

50 "M CHX and 50 "M BFA. The GFP and FM4-64 signals in the epidermal cells are shown in green

and red, respectively. In the merged images, the GFP and FM4-64 overlapping signals appear in

yellow. DMSO was used as a negative control. Arrowheads indicate examples of BOR1-GFP and

FM4-64 (top), and BOR1K590A-GFP and FM4-64 (bottom) co-localization. Bar, 5 "m. B, Distribution

of GFP signals derived from BOR1-GFP or BOR1K590A-GFP in the epidermal cells of the root tips

after treatment with high-B (+B) or –B in the dark (2 h). Arrowhead indicates GFP signals in the

vacuole. Bar, 10 "m. C, Distribution of BOR1-GFP and BOR1K590A-GFP after treatment with

wortmannin (WM) in the epidermal cells of the root tips. The roots were incubated with –B medium

containing 4 "M FM4-64 for 90 min, and then with +B or –B medium containing 16.5 "M WM or

0.0825 % DMSO for 90 min. Arrowheads indicate co-localization. Bar, 60 "m. D, Magnified images

of the boxed areas in (C). Bar, 10 "m.

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A

B

GFP

BOR4

BOR1

BOR1

K590

K689

K649

K649

K689

L468 (BOR1)

E641(BOR1)

L650 (BOR4)

(BOR4)Q475

Pbor1

PBOR1-BOR1-GFP

PBOR1-BOR4-GFP

1kbp

PBOR1-chimera 1-GFP

PBOR1-chimera 2-GFP

N-ter C-ter

i i i i i io o o o o

1 2 3 4 5 6 7 8 9 10

QSTMVGGCVAAMPILKMIPTSVLWGYFAFMAIESLPGNQFWERILLLFTAPSRRFKVLEDY

QSLLVAGAVLAMPAIKLIPTSILWGYFAYMAIDSLPGNQFFERLTLLFVPTSRRFKVLEGA

ETLFDIEKEIDDLLPVEVKEQRVSNLL

ESGFDPEKHLDAYLPVRVNEQRVSNLL

H

H

BOR1

BOR4

443

449

531

537

I

D

GSTTSYPGDLEILDEVMTRSRGEFRHTSSPKVTSSSST

SKRGVQEGDAEILDELTT-SRGELKVRTLN--------

PVNNRSLSQVFSPRVSGIRLGQMSPRVVGNSPKPASCGRSPLNQSSSN

-LNEDKGNQIYPKEKVKAGDGDMSTTRE--------------------

BOR1

BOR4

618

627

704

683

BOR1

BOR4

K590

ATFVETVPFKTIAMFTLFQTTYLLICFGLTWIP

ASFVEKVPYKSMAAFTLLQIFYFGLCYGVTWIP

IAGVMFPLMIMFLIPVRQYLLPRFFKGAHLQDLDAAEYEEAPALPFN---LAAE

VAGIMFPVPFFLLIAIRQYILPKLFNPAHLRELDAAEYEEIPGTPRNPLELSFR

TE

SN

BOR1

BOR4

532

538

617

626

TM10

TM9

chimera 1

BOR1

BOR4

chimera 2

TM-

FIGURE 1

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BO

R1

-GF

P

–BA B+B

BO

R4

-GF

PC

him

era 1

-GF

PC

him

era 2

-GF

P

Ou In

Ou In

Ou In

Ou In

BO

R1

-GF

PB

OR

4-G

FP

Ch

imera 1

-GF

PC

him

era 2

-GF

P

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250

(kDa)

150

100

75

100

75

50

root shoot

0 30

60

120

0 30

60

120

0 30

60

120

(min)

BOR1-GFP

BOR1K590A

-GFPBOR1-GFP

Co

ntr

ol

Co

ntr

ol

A B

C

Ou In

+B4d+B5h

-B +B2h

CBB

T

M

0 30

60

120

BOR1K590A

-GFP

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anti-Ub

anti-GFP

anti-GFP

long-exposure

0 30 60 0 30 60120 0

dm

30 60 120 (min)

BOR1

-GFP

BOR1K590A

-GFP

BOR1K649A,689A

-GFP

250

150

100

75

100

75

100

50

(kDa)

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BFA DMSO

GFP

FM4-64

merged

GFP

FM4-64

merged

GFP mergedFM4-64

GFP mergedFM4-64 GFP mergedFM4-64

BOR1-GFP

BOR1K590A-GFP

BOR1-GFP

BOR1K590A-GFP

BA

C

D

BOR1-GFP

BOR1K590A-GFP

-B+B

-B+B

WM DMSO

GFP mergedFM4-64

-B +B

-B +B

a

a b

b

FIGURE 5

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Koji Kasai, Junpei Takano, Kyoko Miwa, Atsushi Toyoda and Toru FujiwaraArabidopsis thalianatransporter in

High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate

published online December 9, 2010J. Biol. Chem. 

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