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Hereditary Spastic Paraplegia Protein Spartin Is

an FK506-Binding Protein Identified by mRNA Display

Mayuko Tokunaga,

1

Hirokazu Shiheido,

1

Ichigo Hayakawa,

1

Akiko Utsumi,

1

Hideaki Takashima,

1

Nobuhide Doi,

1,

*

Kenichi Horisawa,

1

Yuko Sakuma-Yonemura,

1

Noriko Tabata,

1

and Hiroshi Yanagawa

1

1Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan

*Correspondence:doi@bio.keio.ac.jp

http://dx.doi.org/10.1016/j.chembiol.2013.05.011

SUMMARY

Here, we used mRNA display to search for proteins

that bind to FK506, a potent immunosuppressant

drug, and identified spartin, a hereditary spastic

paraplegia protein, from a human brain cDNA library.

We demonstrated that FK506 binds to the C-terminal

region of spartin and thereby inhibits the interaction

of spartin with TIP47, one of the lipid

droplet-associ-ated proteins. We further confirmed that FK506

inhibits localization of spartin and its binder, an E3

ubiquitin ligase AIP4, in lipid droplets and increases

the protein level of ADRP (adipose

differentiation-related protein), which is a regulator of lipid

homeo-stasis. These results strongly suggest that FK506

suppresses the proteasomal degradation of ADRP,

a substrate of AIP4, by inhibiting the spartin-TIP47

interaction and thereby blocking the localization of

spartin and AIP4 in lipid droplets.

INTRODUCTION

FK506 (tacrolimus) is a potent immunosuppressant drug used for

preventing graft rejection in organ or bone marrow

transplanta-tion and for treating autoimmune diseases, including atopic

dermatitis and rheumatoid arthritis (

Miyata et al., 2005

). FK506

is a 23-membered macrolide lactone that was isolated from

Streptomyces tsukubaensis in 1984 (

Tanaka et al., 1987

). The

molecular mechanism of its immunosuppressive action was

clar-ified in 1991 (

Liu et al., 1991; Schreiber, 1991

). In a normal

immune response, calcineurin dephosphorylates NFAT (nuclear

factor of activated T cells), allowing NFAT to be transferred into

the nucleus, where it enhances the transcription of several

genes, including the cytokine interleukin-2 that serves as a

mas-ter regulator for activation of the immune system (

Emmel et al.,

1989

). Schreiber and colleagues found that FK506 binds to

FKBP12 (FK506 binding protein 12 kDa) and the

FK506-FKBP12 complex inhibits dephosphorylation of NFAT through

binding to calcineurin (

Liu et al., 1991; Schreiber, 1991

).

In this study, we aimed to discover FK506-binding proteins by

using mRNA display (

Nemoto et al., 1997; Roberts and Szostak,

1997

). mRNA display is a method for construction of a library of

protein-mRNA conjugates in which each protein (phenotype) is

covalently linked to its encoding mRNA (genotype) through

puro-mycin (

Nemoto et al., 1997; Roberts and Szostak, 1997

). After

af-finity selection based on the association of a bait (e.g., protein,

nucleic acid, and chemicals) with the protein portion of

conju-gates, the mRNA portion of selected molecules can be easily

amplified by RT-PCR and used for the next round of selection

and ultimately for sequence identification (

Figure 1

). So far,

mRNA display has been used for in vitro selection of proteins

that bind to various targets including proteins (

Horisawa et al.,

2004; Miyamoto-Sato et al., 2005; Tamada et al., 2012

), cis

DNA element (

Tateyama et al., 2006

), and small-molecular

com-pounds (

Shiheido et al., 2012a, 2012b

) from cDNA libraries.

Moreover, mRNA display has already been applied to selection

of FK506-binding proteins from a mixed cDNA library derived

from human liver, kidney, and bone marrow, but only the

FKBP12 gene was preferentially enriched during the PCR

ampli-fication and affinity selection steps (

McPherson et al., 2002

),

probably because FKBP12 is a small protein (

12 kDa) with

high affinity for FK506 (K

d

= 0.4 nM;

Bierer et al., 1990

). Here,

to prevent the preferential enrichment of FKBP12 gene, we

elim-inated short DNA fragments including FKBP12 gene by gel

elec-trophoresis in each round of mRNA display selection.

Further-more, we performed selection of a cDNA library derived from

human brain, because we were most interested in the

neurotox-icity of FK506 (

Shou et al., 1998

). Consequently, we identified

spartin, a hereditary spastic paraplegia (HSP) protein, which is

related to neural disease and disorder (

Bakowska et al., 2008;

Manzini et al., 2010; Patel et al., 2002

), as a FK506 binder and

characterized the effect of FK506 binding on spartin function.

RESULTS

Spartin Interacts with FK506 through Its C-Terminal

Region

Affinity selection of FK506-binding proteins was performed using

FK506-biotin (

Figure 2

A) immobilized on streptavidin beads as

previously described (

McPherson et al., 2002

). After four rounds

of selection, we obtained a C-terminal fragment of SPG20 gene,

encoding spartin (433–584 amino acids;

Figure 2

B) as a distinct

positive clone. No point mutation was found in the fragment.

To determine whether spartin interacts with FK506 directly,

we performed in vitro pull-down assays of green fluorescent

pro-tein (GFP)-fused spartin using FK506-immobilized beads. As

expected, GFP-spartin bound to FK506, while GFP-Spartin

DC,

in which amino acid residues 349–666 of spartin including a

putative FK506 binding region are deleted, did not (

Figure 2

C,

top). This result shows that the C-terminal region of spartin is

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essential for the interaction with FK506. In addition, we

per-formed a competitive pull-down assay in the presence of free

FK506: the amount of GFP-fused spartin bound to biotinylated

FK506 decreased with an increase in free FK506 concentration

(

Figure 2

C, bottom).

Next, we examined whether endogenous spartin binds to

FK506. We performed a pull-down assay of endogenous spartin

in total cell lysates using beads bearing FK506 or an FK506

analog (

Dubowchik et al., 2000

) named FKL that retains the

FKBP12-binding site (

Figure 2

A). The result showed that

endog-enous spartin binds not only to FK506, but also FKL (

Figure 2

D,

top). In addition, we confirmed that the binding of endogenous

spartin with bead-immobilized FK506 was completed with free

FK506 (

Figure 2

D, bottom).

Moreover, we performed a competitive ELISA capable of

quantifying FLAG-tagged spartin bound to immobilized FK506

in the presence of free FK506 or rapamycin (

Figure 2

A). The

amount of FLAG-spartin bound to immobilized FK506 decreased

with an increase in free FK506 or rapamycin concentration (

Fig-ure 2

E). Because FK506, FKL, and rapamycin contain a common

‘‘FKBP12-binding structure’’ (

a-keto proline amide linkage), the

C-terminal region of spartin also probably binds to the

FKBP12-binding portion of FK506.

Finally, we examined the affinity of His

6

-tagged spartin for

bio-tinylated FK506 with surface plasmon resonance (SPR) analysis

(

Figure 2

F). The measured K

d

value was 0.1

mM: the affinity is

quite lower than that for FKBP12 (K

d

= 0.4 nM) but enough to

achieve efficient enrichment within four rounds of mRNA-display

selection.

FK506 Inhibits Spartin Localization in Lipid Droplets

The C-terminal region of spartin is essential for localization in

lipid droplets (

Eastman et al., 2009

). Lipid droplets are cellular

organelles that store neutral lipids, surrounded by a

phospho-lipid monolayer in which certain associated proteins are

embedded. The organelles play a central role in whole-body lipid

homeostasis (

Digel et al., 2010

). On the surface of lipid droplets,

spartin recruits HECT (homologous to the E6-AP carboxyl

termi-nus) E3 ubiquitin ligase AIP4 (atrophin-1-interacting protein 4)

through its N-terminal PPXY motif and thereby promotes

ubiquitination and proteasomal degradation of lipid

droplet-associated protein ADRP (adipose differentiation-related

pro-tein;

Eastman et al., 2009; Edwards et al., 2009; Hooper et al.,

2010

;

Figure 3

A).

First, we examined the effect of FK506 binding to the

C-ter-minal region of spartin on the localization of spartin and AIP4 in

lipid droplets. HeLa cells co-expressing HA-tagged spartin and

FLAG-tagged AIP4 were treated with 0–10

mM FK506 for 1 hr.

Immunofluorescent staining followed by fluorescence

micro-scopy showed that HA-spartin and FLAG-AIP4 were

colocal-ized in lipid droplets in most of the cells without FK506

treat-ment. However, these proteins were colocalized in lipid

droplets in less than half of cells treated with FK506. The

per-centage of lipid droplets in which HA-spartin and FLAG-AIP4

were colocalized decreased from 85% to 24% with 1

mM

and to 14% with 10

mM FK506 (

Figures 3

B and 3C). We also

confirmed that FK506 does not affect the expression level of

either HA-spartin or FLAG-AIP4 (

Figure 3

D). These results

sug-gest that FK506 decreased the amount of spartin and AIP4

proteins localized in lipid droplets not through suppressing

protein expression, but by blocking the localization of the

pro-teins in lipid droplets in an FK506 concentration-dependent

manner.

Next, we examined whether FK506 inhibits the interaction

between spartin and TIP47 (tail interacting protein of 47 kDa),

one of the lipid droplet-associated proteins, because

localiza-tion of spartin in lipid droplets involves interaclocaliza-tion with TIP47

through its C-terminal region (

Eastman et al., 2009

). HA-tagged

spartin and TIP47-GST fusion protein were co-expressed in

HeLa cells, and then the complex of HA-spartin and

TIP47-GST was purified with glutathione beads in the presence of

FK506. We found that the interaction between HA-spartin

and TIP47-GST was inhibited in a FK506

concentration-depen-dent manner (

Figure 3

E). We also performed a competitive

ELISA and found that the amount of T7-tagged spartin bound

to immobilized TIP47-GST decreased with an increase in free

FK506 concentration (

Figure 3

F). This result indicates that

FK506 directly inhibits the interaction between spartin and

TIP47.

Finally, we examined whether FK506 inhibits AIP4-mediated

proteasomal degradation of ADRP because spartin is known to

act as an adaptor that recruits AIP4 to lipid droplets and

in-creases its enzymatic activity (

Hooper et al., 2010

). HeLa cells

were treated with or without 50

mg/ml cycloheximide, a protein

synthesis inhibitor, for 20 min and then treated with 0–20

mM

FK506 for 1 hr. As a result, the ADRP protein level was gradually

increased relative to when the FK506 concentration was

gradu-ally increased, both in the presence and absence of

cyclohexi-mide (

Figure 4

A), while FK506 did not change the ADRP mRNA

level (

Figure 4

B). Furthermore, FK506 did not affect the protein

level of spartin and TIP47 with or without cycloheximide (

Figures

4

C and 4D). These results strongly suggest that FK506

Figure 1. Schematic Representation of mRNA Display

(1) A cDNA library derived from human brain is transcribed and ligated with a PEG-puromycin spacer. (2) The resulting mRNA template is in vitro translated to form a library of protein-mRNA conjugates. (3) The library is incubated with FK506-immobilized beads and unbound molecules are washed away. (4) The bound molecules are eluted with proteinase K and their mRNA portion is amplified by RT-PCR. The resulting DNA is size-fractionated and the larger DNA fragments are used for the next round of selection. (5) After iterative selection, the enriched DNA is analyzed by cloning and sequencing.

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Figure 2. FK506 Bound to the C-Terminal Region of Spartin

(A) The chemical structures of biotinylated FK506, rapamycin, and FKL, which is an FK506 analog that retains the FKBP12-binding site, are depicted. FK506 and FKL were biotinylated through a linker placed outside the FKBP12-binding site.

(B) The domain structure of spartin protein and the mutated region of the SPG20 gene in patients with HSP are shown. Spartin harbors an N-terminal MIT (microtubule interacting and trafficking) domain, a PPXY motif for WW domain recognition, and a less well-characterized plant-related senescence domain at its C terminus. Patients with HSP have a two-base-pair deletion (364_365 delAT) or a single-base-pair deletion (1110 delA) in the spartin gene, resulting in a frameshift and C-terminal truncation of spartin (Manzini et al., 2010; Patel et al., 2002). The C-terminal region (433–584 amino acids) of spartin (the FK506 binding region) was selected by using mRNA display.

(C, top) Pull-down assays of GFP-fused spartin with FK506. HEK293T cells were transfected with a construct expressing GFP-spartin, GFP-SpartinDC (a C-terminal deletion mutant of spartin, 1–348 amino acids) or GFP. After 48 hr, whole cell lysates were prepared and used for pull-down assays with FK506-immobilized (+) or nonFK506-immobilized () beads. Each fraction was separated with 8% SDS-PAGE, and the fluorescence of GFP was detected by using Molecular Imager FX. A band, which shifted 10 kDa upward (marked with an asterisk), presumably represents the monoubiquitinated form of spartin (Bakowska et al., 2007). I, input; F, flow through; B, beads fraction. (Bottom) Competitive pull-down assays of GFP-fused spartin with FK506-immobilized beads in the presence of 0–100mM free FK506.

(D, top) Pull-down assays of HeLa cell endogenous spartin with FK506 and FKL. Whole cell lysates of HeLa cells were prepared and pull-down assays were performed. Each fraction was separated by 8% SDS-PAGE and bands were detected with western blot analysis with an antibody against spartin. (Bottom) Competitive pull-down assays of HeLa cell endogenous spartin with FK506-immobilized beads were performed in the presence of 0–100mM free FK506 as described above. Mock: beads without any compound.

(E) FLAG-spartin was generated by in vitro translation, bound to biotinylated FK506 in the presence of various concentrations of free FK506 or rapamycin and quantified with ELISA. Error bars represent SD.

(F) A representative biosensorgram of spartin binding on SA sensor chips with immobilized biotinylated-FK506 is shown. To determine the Kdvalues, three

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suppresses the proteasomal degradation of ADRP, a substrate

of AIP4, by blocking the localization of spartin in lipid droplets

and the enhancement of the ubiquitin ligase activity of AIP4.

Figure 3. FK506 Inhibited Localization of Spartin and AIP4 in Lipid Droplets

(A) A hypothesis: FK506 binds to the C terminus of spartin and inhibits its binding to lipid droplet-associated protein TIP47. Thereby, it prevents colocalization of spartin and AIP4 in lipid droplets and the proteasomal degradation of ADRP. The colors of these proteins refer to the fluorescence colors inFigure 3B.

(B) Immunofluorescence staining using anti-HA-tag (CF640, red) and anti-AIP4 (Alexa568, blue) of HeLa cells transfected with HA-spartin and FLAG-AIP4 genes and, after 24 hr, treated with 0–10mM FK506 for 1 hr. Lipid droplet staining with BOD-IPY493/503 (green) is also shown. The samples were observed under a fluorescence microscope. Lipid droplets colocalized with HA-spartin and FLAG-AIP4 are indicated by white arrowheads, otherwise by a white arrow. Bar = 10mm. (C) Colocalization of HA-spartin and FLAG-AIP4 in lipid droplets was scored for 40–90 lipid droplets in each of three independent experiments. The ratio under the indicated conditions is shown. Error bars represent SD.

(D) Expression levels of HA-spartin and FLAG-AIP4 in HeLa cells treated with 0–10mM FK506 were analyzed with western blot analysis with antibody against HA-tag and FLAG-tag, respec-tively.b-Actin is shown to verify equal loading. (E) GST pull-down assays in the presence of 0–20mM FK506 using HA-spartin and TIP47-GST co-expressed in HEK293T cells. Each fraction was separated with 8% SDS-PAGE and then analyzed with western blot analysis with an antibody against HA-tag or GST. I, input; B, beads fraction. (F) T7-tagged spartin was generated by in vitro translation, bound to TIP47-GST in the presence of various concentrations of free FK506, and quantified with ELISA. Error bars represent SD.

DISCUSSION

There are several possible reasons why

spartin was successfully selected from

the human brain cDNA library, while in

the previous study only FKBP12 was

selected from human liver, kidney, and

bone marrow cDNA libraries by mRNA

display selection using identical

FK506-biotin as bait (

McPherson et al., 2002

).

One possibility is that spartin is highly

ex-pressed in human brain, but not in liver,

kidney, and bone marrow, and so would

be identified only when a human brain

cDNA library was used. However, this is

not the case, because the expression

levels in these organs are reported to be

similar to those in brain (

Manzini et al.,

2010

). Therefore, we thought that our

use of DNA size fractionation was the reason for the apparent

discrepancy. Because the FK506-binding affinity of FKBP12

(K

d

= 0.4 nM;

Bierer et al., 1990

) is quite higher than that of spartin

(5)

(0.1

mM), the FKBP12 gene was preferentially enriched during

affinity selection in the previous study (

McPherson et al., 2002

).

Because we eliminated DNA fragments shorter than

400 bp

with gel electrophoresis, the C-terminal fragment of the spartin

gene (433–584 amino acids;

450 bp) was selected, whereas

no FKBP12 gene (

330 bp) was identified from our selection. If

this modification of the mRNA display procedure were adopted

for selection with cDNA libraries derived from other organs

such as liver and kidney, we believe that other FK506-binding

proteins might be discovered.

Because

the

affinity

of

the

spartin-FK506

interaction

(K

d

= 0.1

mM) is quite lower than that of the FK506-FKBP12

inter-action (0.4 nM), one may question whether this interinter-action is

physiologically relevant. Although further studies are needed,

the affinity of the known spartin-AIP4 interaction (K

d

= 0.34

mM;

Hooper et al., 2010

) is even lower than that of the

spartin-FK506 interaction, and thus, we believe that the endogenous

concentration of spartin is enough to bind to other molecules

with submicromolar affinity.

Spartin is related to neural disease and disorder. Two different

mutations (364_365 delAT or 1110 delA) that result in premature

truncation of the C-terminal region of spartin (

Figure 2

B) are

associated with hereditary spastic paraplegia (HSP), an inherited

neurologic disease characterized by paraparesis, spasticity, and

muscle wasting in the lower limbs (

Bakowska et al., 2008;

Manzini et al., 2010; Patel et al., 2002

). On the other hand,

neuro-toxicity, including disturbed consciousness, neuropathy, and

CNS damage, is a side effect of FK506 (

Shou et al., 1998

). Since

Brasaemle and colleagues reported that ADRP regulates the

turnover of lipid droplets and its expression level decreases as

adipocytes differentiate (

Brasaemle et al., 1997

), spartin is

impli-cated in regulation of the turnover of lipids through ADRP.

Aber-rant lipid droplet morphogenesis and lipid metabolism can

apparently lead to axonal damage. For example, seipin, one of

the HSP-associated proteins, like spartin, causes lipodystrophy

with abnormal lipid metabolism and is suggested to cause motor

neuron disease through the accumulation of lipids in motor

neu-rons (

Agarwal and Garg, 2004

). Additionally, abnormal lipid

metabolism affects the integrity of nervous system myelin

(

Chrast et al., 2011

). Furthermore, FK506 frequently induces

myopathy in kidney transplantation patients (

Guis et al., 2003

),

and lipid droplet-associated proteins such as PNPLA2 were

re-ported to induce myopathy in neutral lipid storage disease

(

Fischer et al., 2007

). These previous reports are not inconsistent

with the idea that FK506 induces at least some of its side effects,

including neurologic disorders and myopathy, by inducing

abnormal lipid metabolism as a result of modulating the protein

level of ADRP, although the precise mechanisms remain to be

fully established.

SIGNIFICANCE

The identification of proteins that bind to a drug of interest is

likely to be an effective approach to elucidate potential side

effects and their mechanisms or unknown indications. In

this study, we used mRNA display to identify proteins as

binders to FK506, a potent immunosuppressant drug. We

identified a FK506-binding protein, spartin, which is related

to neural disease and disorder. We demonstrated that

FK506 binds to the C-terminal region of spartin and thereby

inhibits the interaction of spartin with TIP47, one of the lipid

droplet-associated proteins. We further confirmed that

FK506 inhibits colocalization of spartin and AIP4 in lipid

droplets, which increases the protein level of ADRP, a

regu-lator of lipid homeostasis. These results strongly suggest

that FK506 suppresses the proteasomal degradation of

ADRP, a substrate of AIP4, by blocking the localization of

AIP4 in lipid droplets. As shown in this study, mRNA display

is useful for the identification and characterization of

drug-protein interactions.

Figure 4. FK506 Inhibited Proteasomal Degradation of ADRP (A, C, and D) Expression level of ADRP (A), spartin (C), and TIP47 (D) protein in HeLa cells treated with (+) or without () cycloheximide, and then treated with 0–20mM FK506 for 1 hr. The whole cell lysates were separated with 8% SDS-PAGE and analyzed with western blot analysis with an antibody against ADRP, spartin, and TIP47, respectively (top). The band intensity was quantified and normalized with that ofb-actin (bottom). Error bars represent SD. (B) The amount of ADRP mRNA in HeLa cells treated with 0 or 20mM FK506 for 1 hr. The total RNA was extracted and then amplified by RT-PCR with specific primer for GAPDH or ADRP.

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EXPERIMENTAL PROCEDURES

mRNA Display Selection for FK506-Binding Proteins

Construction of the mRNA-displayed protein library was performed as previ-ously described (Doi et al., 2007; Horisawa et al., 2004). Briefly, a human brain cDNA library was amplified, transcribed, ligated with a PEG-puromycin spacer, p(dCp)2-T(fluorescein)p-PEGp-(dCp)2-puromycin (Miyamoto-Sato

et al., 2003), and translated in a cell-free wheat germ translation system (Prom-ega). The resulting mRNA-displayed protein library was added to FK506-im-mobilized beads, mixed for 1 hr at 4C, washed five times, and then eluted with proteinase K (Takara). The supernatant was purified and amplified with RT-PCR. The RT-PCR product was separated by 2% agarose gel electropho-resis. A fraction of the gel including DNA fragments longer than 400 bp was packed with TAE buffer in seamless cellulose tubing (Eidia, Ibaraki, Japan) and eluted by electrophoresis at 100 V for 2 hr. The extracted DNA was ampli-fied, puriampli-fied, and used for the next round selection. After several rounds of selection, the RT-PCR product was cloned and sequenced. Details are pro-vided in theSupplemental Experimental Procedures.

DNA Construction

The following plasmids and PCR products were prepared as described in the Supplemental Experimental Procedures: pQBI25-SPG20 containing SPG20 gene and its deletion mutant pQBI25-SPG20DC; pcDNA3.1/Hygro()-HA-SPG20 containing HA-tagged pcDNA3.1/Hygro()-HA-SPG20 gene and its deletion mutant cDNA3.1/Hygro()-HA-SPG201110delA; the PCR product

SP6-O29-FLAG-SPG20-His-polyA containing FLAG-tagged SPG20 gene; pCR3.3TOPO-SPG20 containing His6-tagged SPG20 gene; pCMV-tag2A-AIP4 containing

FLAG-tagged AIP4 gene; pcDNA3.1/Hygro()-TIP47-GST containing GST-fused TIP47 gene; and the PCR product, SP6-O29-TIP47-GST-His-polyA.

Cell Lines

The human embryonic kidney cell line HEK293T (RIKEN Cell Bank, Ibaraki, Japan, 2002) and human cervical cancer cell line HeLa (RIKEN Cell Bank, Ibar-aki, Japan, 2002) were maintained in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) with 10% fetal bovine serum and 1% penicillin and streptomycin.

Western Blotting

HeLa cells cultured to 80% confluence were treated with 0–100mM FK506 for 1–48 hr. The cells were rinsed with PBS followed by lysis with RIPA buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycho-late, and 1% NP-40, 0.05% SDS) containing a protease inhibitor cocktail (Nacalai Tesque) and a phosphatase inhibitor cocktail (Nacalai Tesque) so that protein phosphorylation could be examined. For observation of the ADRP expression level, HeLa cells were treated with 50mg/ml cycloheximide (Sigma, St. Louis, MO, USA) before treating with FK506. To examine the expression level of spartin and AIP4, lysates were homogenized by sonication for 10 min. Lysates were then centrifuged at 4C, 15,000 rpm for 15 min, and the supernatant was removed. Protein concentrations were determined using a BCA protein assay kit (Thermo, Rockford, IL, USA) and an equivalent amount of protein was electrophoresed on 8% SDS-PAGE gel. Bands were detected with primary antibodies against spartin (Santa Cruz, Santa Cruz, CA, USA), HA-tag (Cell Signaling Technology, Beverly, MA, USA), FLAG-tag,b-actin, GST (Sigma), AIP4, ADRP (Abcam, Cambridge, MA, USA) or TIP47 (Novus Biologicals, Littleton, CO, USA), and HRP-conjugated secondary antibody against rabbit or mouse (Millipore, Billerica, MA, USA). The blots were devel-oped using ECL chemi-luminescence reagents (GE Healthcare, Waukesha, WI, USA).

FK506-Spartin Binding Assay

HEK293T cells were transfected with plasmid SPG20 or pQBI25-SPG20DC using Lipofectamine 2000 (Invitrogen). After 48 hr, these cells were rinsed with PBS followed by lysis with lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, and 0.1% Tween 20) containing the protease inhibitor cocktail. The cell lysates were then centrifuged at 12,0003 g for 20 min at 4C. The supernatant was mixed with 10% EtOH and added to FK506-immobilized beads on a rotator for 1 hr at 4C. To perform the compet-itive binding assay, 0–100mM FK506 was added to the beads with EtOH and

the supernatant. The beads were washed with lysis buffer containing 10% EtOH, resuspended in SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% sodium dodecylsulfate, 10% sucrose, and 0.01% bromophenol blue), and vor-texed to elute bound molecules. The resulting eluate was separated with 8% SDS-PAGE and the fluorescence of the GFP tag was detected with a Molec-ular Imager FX (BioRad, Richmond, CA, USA). For endogenous spartin, cell lysates from HeLa cells were used. The resulting eluate was separated with 8% SDS-PAGE and bands were detected with western blotting.

Surface Plasmon Resonance Analysis

HEK293T cells were transfected with plasmid pCR3.3TOPO-SPG20 using Lip-ofectamine 2000. After 24 hr, these cells were rinsed with PBS followed by lysis with IPP150 (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% NP-40) contain-ing the protease inhibitor cocktail. The cell lysates were then homogenized with sonication followed by centrifugation at 12,0003 g for 15 min at 4C.

The supernatant was removed and added to TALON Metal Affinity beads (Nacalai Tesque) pre-equilibrated with IPP150 on a rotator for overnight at 4C. The resulting beads were washed with IPP150 containing 0 or 20 mM imidazole and the protein was eluted with IPP150 containing 500 mM imidazole.

The resulting eluates were further purified with gel filtration chromatography with AKTA (GE Healthcare). We used the Superose 12 column (GE Healthcare) and a mixture of 0.1 mg/ml blue dextran 2000, 0.35 mg/ml albumin, 0.35 mg/ml ovalbumin, 0.35 mg/ml chymotrypsin A, and 0.5 ribonuclease A as a molecular weight marker.

Binding kinetics were determined with SPR analysis with a Biacore 3000 (GE Healthcare). All experiments were performed at 25C using HBS-EP buffer. Biotinylated FK506 was immobilized onto the SA sensor chip (GE Healthcare). The measurements were performed under conditions of 388.5 resonance units of the ligand and at a flow rate of 10ml/min. To determine dissociation con-stants, three different concentrations of purified spartin were injected. The injection period for association was 350 s. After each measurement, the chip surface was regenerated with 10 ml of Glycine 2.0 (GE Healthcare). The bind-ing data were analyzed with the steady-state affinity model in the BIAevalua-tion software ver. 4.1 (GE Healthcare).

ELISA

An FK506-immobilized plate was prepared by adding 1,200 nM biotinylated FK506 in TBST (TBS with 0.1% Tween 20) to a Streptavidin C8 Transparent plate (Nunc, Roskilde, Denmark) and shaking it for 1 hr at room temperature. The plate was washed with TBST and nonspecific binding was blocked with Block Buffer (DIG wash and block buffer set, Roche) by shaking for 1 hr at room temperature. SP6-O29-T7-FLAG-spartin-His-polyA, SP6-O29-T7-FLAG-FKBP12-His-polyA and SP6-O29-T7-CBPzz-polyA were transcribed using RiboMax large-scale RNA production systems-SP6, and the transcrip-tion product was purified with an RNeasy mini kit. The resulting mRNA was translated in a cell-free wheat germ translation system. In vitro translated T7-FLAG-spartin protein mixed with 0–100mM FK506 or rapamycin (Sigma) in TBST was added to the wells and the plate was shaken for 1 hr at room temperature. It was washed with TBST, followed by incubation with HRP-con-jugated antibody against FLAG-tag (Sigma) in TBST for 1 hr at room tempera-ture. The plate was washed once more and the binding amount of each protein was measured by using the ELISA POD substrate TMB kit (Nacalai Tesque) with a SAFIRE microplate reader (Tecan, Mannendorf, Switzerland).

Similarly, SP6-O29-T7-FLAG-spartin-His-polyA and SP6-O29-FLAG-TIP47-GST-His-polyA were transcribed using RiboMax large-scale RNA pro-duction systems-SP6 and the transcription product was purified with an RNeasy mini kit. The resulting mRNA was translated in a cell-free wheat germ translation system. The TIP47-GST immobilized plate was prepared by adding the in vitro translated TIP47-GST protein to a Glutathione F96 clear plate (Nunc) and shaking it for 1 hr at room temperature. In vitro translated T7-tagged spartin protein mixed with 0–100mM FK506 in TBST was added to the wells and the plate was shaken for 1 hr at room temperature. It was washed with TBST, followed by incubation with HRP-conjugated antibody against T7-tag (Novagen) in TBST for 1 hr at room temperature. The plate was washed once more, and the binding amount of each protein was measured by using the ELISA POD substrate TMB kit with a SAFIRE micro-plate reader.

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Immunofluorescence

HeLa cells cultured to 80% confluence on poly-L-lysine-coated cover gla-sses (Matsunami Glass, Osaka, Japan) were transfected with plasmids pcDNA3.1/Hygro()-HA-spartin and pCMV-tag2A-AIP4 using the GeneJuice Transfection Reagent. After 24 hr, the transfected cells were treated with 500mM oleic acid (Nacalai Tesque) and 1 mM FK506 for 1 hr. These cells were then fixed with 4% paraformaldehyde for 20 min followed by permeabi-lization with 0.1% saponin (Nacalai Tesque) for 10 min. The sample was stained with antibody against the HA tag and AIP4, followed by CF640R-con-jugated anti-mouse IgG (Biotium, Hayward, CA, USA) and Alexa568-conju-gated anti-rabbit IgG (Invitrogen). Lipids were stained with BODIPY493/503 (Invitrogen). Photobleaching was prevented by using the Slow Fade gold anti-fade reagent with DAPI (Invitrogen).

Coimmunoprecipitation

The cell lysates from HEK293T cells transfected with pcDNA3.1/Hygro()-HA-SPG20 and pCMV-tag2A-AIP4 was added to anti-FLAG M2 agarose beads (Nacalai Tesque) that had been pre-equilibrated with lysis buffer three times for 2 hr on a rotator at 4C. The resulting beads were washed with lysis buffer three times and resuspended in SDS sample buffer containing 2% b-mercap-toethanol, followed by heating for 10 min at 95C to denature proteins. The su-pernatant was separated by 8% SDS-PAGE and analyzed with western blotting.

GST Pull-Down Assays

HEK293T cells were transfected with plasmids pcDNA3.1/Hygro( )-HA-spar-tin and pcDNA3.1/Hygro()-TIP47-GST using the GeneJuice Transfection Reagent. After 48 hr, the transfected cells were rinsed with PBS and lysed with GST lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 5% glycerol) followed by centrifugation at 12,0003 g for 15 min at 4C, and then the supernatant was collected. The supernatant was mixed with 0–20mM FK506 followed by incubation with Glutathione Sepharose 4B (GE Healthcare) that had been pre-equilibrated with GST wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100). The resulting beads were washed three times with GST wash buffer and resuspended in SDS sample buffer containing 2% b-mercaptoetha-nol, followed by heating for 10 min at 95C. The resulting eluate was separated by 8% SDS-PAGE and analyzed with western blotting.

RT-PCR Analysis

Details are provided in theSupplemental Experimental Procedures.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j. chembiol.2013.05.011.

ACKNOWLEDGMENTS

We thank Drs. Seiji Tateyama and Toru Tsuji for experimental advice and useful discussions. This research was supported in part by the Program for Promo-tion of Fundamental Studies in Health Sciences of the NIBIO of Japan; a grant for the Core Research for Evolutionary Science and Technology of the Japan Science and Technology Agency; and a grant-in-aid for scientific research, a special coordination fund grant, and a Strategic Research Foundation Grant-aided Project for Private Universities (S0801008) from the MEXT of Japan. Received: May 15, 2012 Revised: May 22, 2013 Accepted: May 24, 2013 Published: July 25, 2013 REFERENCES

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