Synthetic Ubiquinones Specifically Bind to Mitochondrial Voltage

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Synthetic Ubiquinones Specifically Bind to Mitochondrial Voltage-Dependent Anion Channel 1 (VDAC1) in Saccharomyces cerevisiae Mitochondria Masatoshi Murai, Ayaka Okuda, Takenori Yamamoto, Yasuo Shinohara, and Hideto Miyoshi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01011 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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Synthetic Ubiquinones Specifically Bind to Mitochondrial VoltageDependent Anion Channel 1 (VDAC1) in Saccharomyces cerevisiae Mitochondria

Masatoshi Murai 1 , Ayaka Okuda 1 , Takenori Yamamoto 2 , Yasuo Shinohara 2 , and Hideto Miyoshi 1*

1

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,

Sakyo-ku, Kyoto 606-8502, Japan, and 2 Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan.

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ABSTRACT The role of voltage-dependent anion channel (VDAC) as a metabolic gate of the mitochondrial outer membrane has been firmly established; however, its involvement in the regulation of mitochondrial permeability transition (PT) remains extremely controversial.

Although some low-molecular-weight chemicals have been proposed to

modulate the regulatory role of VDAC in the induction of PT, direct binding between these chemicals and VDAC has not yet been demonstrated.

In the present study, we

investigated whether the ubiquinone molecule directly binds to VDAC in Saccharomyces cerevisiae mitochondria through a photoaffinity labeling technique using two photoreactive ubiquinones (PUQ-1 and PUQ-2).

The results of the labeling experiments

demonstrated that PUQ-1 and PUQ-2 specifically bind to VDAC1 and that the labeled position is located in the C-terminal region Phe221–Lys234, connecting the 15th and 16th β-strand sheets.

Mutations introduced in this region (R224A, Y225A, D228A, and

Y225A/D228A) hardly affected the binding affinity of PUQ-1.

PUQ-1 and PUQ-2 both

significantly suppressed the Ca 2+ -induced mitochondrial PT (monitored by mitochondrial swelling) at the one digit µM level.

Thus, the results of the present study provided, for

the first time to our knowledge, direct evidence indicating that the ubiquinone molecule specifically binds to VDAC1 through its quinone-head ring.


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ABBREVIATIONS CBB, Coomassie brilliant blue R250; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; MS, mass spectrometry; PT, permeability transition; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; UQ, ubiquinone; UQ 0 , ubiquinone-0, UQ 2 , ubiquinone-2; VDAC, voltage-dependent anion channel.


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INTRODUCTION Mitochondrial voltage-dependent anion channel (VDAC) is a mitochondrial outer membrane channel formed by a single polypeptide with a molecular mass of 32 kDa (1, 2).

This protein facilitates the trafficking of ions and metabolites across the

mitochondrial outer membrane, thereby regulating communication between mitochondria and the cytosol.

According to structural models of various VDACs determined by NMR

spectroscopy (3, 4) and X-ray crystallographic study (4, 5), this protein has 19-stranded β-sheets, which form an integral β-barrel structure with an open diameter of approximately 25Å, and an N-terminal α-helical segment that transverses the pore, and may be critical for channel gating (3-6).

Extensive electrophysiological studies

revealed that VDAC has two different conformational states the so called open- and closed-states, which elicit different conductance depending on membrane potential differences (1, 2, 6). VDAC has been identified as a key player in mitochondria-meditated apoptosis via interactions with pro-apoptotic members of Bcl-2 family proteins and hexokinases, both of which increase the permeability of the outer membrane and release of apoptogenic proteins such as cytochrome c from mitochondria (7, 8).

Although it has not yet been

established whether VDAC functions as a monomer or oligomers, the assembly of homooligomers may be critical for allowing the passage of cytochrome c, which is larger than the diameter of VDAC pore (8).

Since VDAC regulates the uptake of important ions

and metabolites such as Ca 2+ , phosphocreatine, and nucleotides, prolonged VDAC closure may serve as a pro-apoptotic signal (1, 8). Another model of mitochondria-meditated cell death is the induction of mitochondrial permeability transition (PT), which is now considered to be associated with ischemia/reperfusion injury in the heart and brain (9-11).

Mitochondrial PT was

originally discovered as an in vitro event that is triggered by the accumulation of Ca 2+ in the matrix or oxidative stress, and is followed by a non-specific increase in the permeability of the mitochondrial inner membrane, ultimately resulting in mitochondrial swelling and the rupture of the outer membrane (10).

VDAC, along with a ADP/ATP

carrier (AAC), phosphate carrier (PiC), cyclophilin D, translocator protein of the outer 4 ACS Paragon Plus Environment

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membrane (TSPO), and ATP synthase have been proposed to form an mitochondrial permeability transition pore (PTP) complex (or the so-called ATP synthasome), which spans the outer and inner mitochondrial membranes and permeabilizes small molecules with molecular mass up to ~1.5 kDa (8, 11).

However, since the deletion of a specific

candidate of the PTP complex, such as VDAC (12), AAC (13), and TSPO (14), was previously shown to not inhibit the induction of PT, the molecular composition of the PTP complex remains controversial, and it currently remains unclear whether VDAC is involved in the regulation of PT (10, 11, 15, 16). Previous studies demonstrated that VDAC interacts with various low-molecularweight chemicals, which have been shown to inhibit PTP opening or regulate apoptotic pathways (17-19).

Of particular interest is the inclusion of short-chain ubiquinones

(UQs) in these chemicals.

Fontaine et al. reported that ubiquinone-0 (UQ 0 ) markedly

inhibits the PT induced by Ca 2+ overload with isolated rat liver mitochondria (20). Although the regulatory effects of UQs (both inductive and inhibitory) on mitochondrial PT has been investigated since the late 1990s using various short-chain UQs (21-23), the molecular target of UQs remains to be identified.

Cesura et al. showed that a PT

inhibitor Ro 68-3400 specifically binds to a protein, the apparent molecular mass of which corresponds to that of VDAC, and the binding was suppressed in the presence of UQ 0 , suggesting that VDAC is the target of Ro 68-3400 (24).

However, this notion was

later revised because Ro 68-3400 bound to some protein (not identified), even in mitochondria isolated from VDAC knock-out mouse (25).

Leung et al. demonstrated

that a PT accelerator phenylarsine oxide (a vicinal thiol reagent) modifies AAC and PiC, and these modifications are suppressed in the presence of UQ 0 or Ro 68-3400 (26).

Thus,

to the best of our knowledge, direct binding between UQs and specific mitochondrial protein(s) supposed to be component of the PTP complex has not yet been confirmed. In an attempt to clarify whether the ubiquinone molecule directly binds to VDAC, we herein performed the photoaffinity labeling experiments using photoreactive UQ derivatives

(PUQ-1

and

Saccharomyces cerevisiae.

PUQ-2,

Figure

1)

with

isolated

mitochondria

from

By means of careful biochemical and proteomic analyses,

we revealed that the quinone-head ring of photoreactive UQs specifically binds to the C5 ACS Paragon Plus Environment

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terminal region Phe221-Lys234 in VDAC1, connecting the 15th and 16th β-strand sheets. The labeling of VDAC1 was completely suppressed by an excess amount of other shortchain UQs such as UQ 0 and PUQ-2.

PUQ-1 and PUQ-2 suppressed the Ca 2+ -induced

mitochondrial PT at the one digit µM level, suggesting that the bound quinone would modulate the regulatory role of VDAC1 in the induction of PT.

The results of the

present study demonstrate, for the first time, that the ubiquinone molecule specifically binds to VDAC1 through the quinone-head ring.


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EXPERIMENTAL PROCEDURES Materials PUQ-1 is the same sample as used previously (27). by Eisai Co., Ltd. (Tokyo Japan).

UQ 2 was generously provided

UQ 0 , cholesterol, and cyclosporine A were purchased

from Tokyo Chemical Industry (Tokyo, Japan) and Wako Pure Chemicals (Osaka, Japan), respectively.

Monoclonal antibody for S. cerevisiae VDAC1 was purchased from

MitoSciences (Eugene, OR). (Hercules, CA).

Protein standards for SDS-PAGE were from Bio-Rad

Other reagents were of analytical grade.

Yeast culture and isolation of mitochondria from S. cerevisiae The haploid strains of S. cerevisiae used in this study were W303-1A (MATa ade2-1 leu2-3,112 his3-22,15 trp1-1 ura3-1 can1-100), W303-1B (MATα ade2-1 leu2-3,112 his3-22,15 trp1-1 ura3-1 can1-100), and Δpor1 (MATα ade2-1 leu2-3,112 his3-22,15 trp1-1 ura3-1 can1-100 por1::HIS3) (28).

Yeast mitochondria were isolated according

to the procedures by Glick and Pon (29) with minor modifications.

Briefly, yeast cells

were grown in 10 mL (5 mL x 2 test tubes) of semisynthetic lactate medium (2% lactic acid, 0.3% yeast extract, 0.05% glucose, 0.05% CaCl 2 , 0.05% NaCl, 0.06% MgCl 2 , 0.1% KH 2 PO 4 , 0.1% NH 4 Cl) or YP medium supplemented with galactose (1% yeast extract, 2% peptone, 2% galactose) at 28˚C for 36 h with rotation at 250 rpm.

The pre-culture

was used to inoculate 800 mL (400 mL x 2 flasks) of the same medium and cells were grown at 120 rpm at 28 °C until A 600 reached 2–4. Yeast cells were centrifuged at 2,500 x g for 5 min, washed with distilled water, and reduced in buffer containing 100 mM Tris/SO 4 and 10 mM DTT (pH 8.0) at 28˚C for 15 min at concentration of 2 mL/g of the wet weight of the cells.

They were centrifuged at

1,400 x g for 5 min, washed with distilled water, and resuspended in digestion buffer containing 1.2 M sorbitol and 20 mM KPi (pH 7.4) at 6.7 mL/g of the wet weight of the cells.

Zymolyase 20T (Nacalai-tesque, Kyoto, Japan) was added at 1.7 mg/mL, and the

suspension was incubated at 28˚C for 30 min with gentle agitation. The spheroplasts obtained were pelleted at 1,400 x g for 5 min, washed in buffer containing 1.2 M sorbitol and 20 mM KPi (pH 7.4), and resuspended in lysis buffer 7 ACS Paragon Plus Environment

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containing 0.6 M mannitol, 10 mM Tris/HCl (pH 7.4), 0.1 mM EDTA, 0.1% BSA, and protease inhibitor cocktail (Sigma) at 6.7 mL/g of the wet weight of the cells.

After

careful homogenization by 5 strokes with a loose-fit Teflon homogenizer, homogenates were centrifuged twice at 800 x g for 5 min. x g for 10 min. same buffer.

The supernatant was centrifuged at 6,800

The final mitochondrial pellet was resuspended in a small volume of the Typically, 2–3 mg of mitochondria was obtained from 800 mL culture.

Construction of the expression vector pRS314 (YCp type containing TRP1) was used as a vector for the transformation of POR1 mutants.

This vector had a constitutive promoter (30).

The promoter region of

the POR1 gene, an upper region of approximately 560 bp of the POR1 gene on the yeast genome, was obtained by PCR to yeast genome DNA as a template (31).

After the

creation of a new EcoRI site at its 5’-terminus and NdeI site at its 3’-terminus, the promoter fragment was subcloned into the EcoRI and NdeI sites of pRS314.

This

expression plasmid is referred to as pRS314-P1P. Preparation of expression plasmids for POR1 mutants The DNA fragment encoding POR1 was prepared by PCR using the primers GE2266 and GE2267.

The nucleotide sequence of POR1 in the database was NM_001182894.

The DNA fragments encoding the POR1 mutants were prepared using the overlapextension PCR method eith the mutagenic primers listed in Supplemental Table S2. Prepared DNA fragments were subcloned into the NdeI and BamHI sites of pRS314-P1P, and the sequences of DNAs encoding these mutants were confirmed.

For expression of

these POR1 mutants, the prepared expression plasmids were introduced into the por1disrupted yeast strain (MATα ade2-1 leu2-3,112 his3-22,15 trp1-1 ura3-1 can1-100 por1::HIS3) (28).

All recombinant DNA experiments were performed according to the

guidelines of the University of Tokushima (approval number: 25-310). Photoaffinity labeling Yeast mitochondria (0.6 mg/mL, 100–500 µL) were resuspended in buffer containing 8 ACS Paragon Plus Environment

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0.45 M sorbitol, 20 mM Tris/HCl, 0.5 mM EDTA, 5 mM MgCl 2 , and 10 mM K 2 PO 4 (pH 6.8), and incubated with PUQ-1 or PUQ-2 (1–10 µM) in the presence of 50 µM NADH for 10 min.

Samples were photo-irradiated with a long wavelength UV lamp (Black Ray

model B-100A, Upland, CA) on ice for 10 min, positioned 10 cm from the light source. Labeled mitochondria were collected by centrifugation (15,000 rpm, 5 min) and solubilized in buffer containing 50 mM Tris/HCl (pH 8.0) and 1% (w/v) SDS.

The

proteins labeled by PUQ-1 (or PUQ-2) were covalently conjugated with the fluorescent TAMRA–azido or biotin–SS–azido tag (Figure 1) via click chemistry using the Click-iT protein reaction buffer kit (Life Technologies, Carlsbad, CA) according to the manufacture’s

instructions.

Proteins

were

recovered

by

methanol/chloroform

precipitation, and subjected to further analyses. Partial purification of VDAC1 by hydroxyapatite chromatography Yeast

mitochondrial

chromatography (28, 32).

VDAC1

was

partially

purified

using

hydroxyapatite

Mitochondria (100–500 µg of protein) labeled by PUQ-1 (or

PUQ-2) were solubilized in buffer (50 µL) containing 10 mM KPi, 20 mM KCl, 1.0 mM EDTA, 0.1 mM PMSF, and 2% Triton X-100 (pH 7.2) on ice for 1 h. were removed by centrifugation at 15,000 rpm for 5 min.

Insoluble materials

The supernatant was loaded

onto a small hydroxyapatite column (150–200 µL volume) equilibrated with the same buffer containing 1% Triton X-100.

The flow-through fraction (200 µL) was collected,

treated with methanol/chloroform, and the resulting precipitate was subjected to conjugation with a fluorescent TAMRA–azido tag. Electrophoresis Proteins conjugated with the fluorescent TAMRA tag were separated on a Laemmlitype SDS gel (33).

The migration pattern of fluorescent proteins was visualized by the

model FLA-5100 Bio-imaging analyzer (Fuji Film, Tokyo, Japan) or Typhoon FLA9500 (GE Healthcare, Buckinghamshire, UK) using a 532 nm light source and LPG (575 nm) filter.

Data processing was conducted using Multi Gauge software (Fuji Film) or Image

Quant, respectively. 9 ACS Paragon Plus Environment

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Biotinylated proteins were subjected to two-dimensional gel electrophoresis (IEF/SDS-PAGE) according to the same conditions described previously (34) using the IPGphor system (GE Healthcare) with an Immobiline Dry Strip (7 cm, pH 3-10, GE Healthcare).

The resolved proteins were transferred onto an PVDF membrane, followed

by blocking with 1% gelatin and incubation with AP-conjugated streptavidin (GE Healthcare), as described elsewhere (27).

The membrane was developed with

NBT/BCIP colorimetric reagents (Bio-rad). Enrichment of the labeled protein To enrich the labeled protein, mitochondria were labeled by 5 µM PUQ-1 (or PUQ2), denatured in Tris-buffered saline (TBS) containing 2% (w/v) SDS, and conjugated with a cleavable biotin–SS–azido (Figure 1) via Cu + -catalyzed click chemistry (27). The biotinylated protein was captured on streptavidin-agarose (Sigma) in TBS buffer containing 1% (w/v) Triton X-100, and released in Laemmli-type SDS-PAGE sample buffer containing 2.5% mercaptoethanol.

Capture/release procedures were monitored

by a yeast VDAC antibody (1:3000 dilution, Abcam) as described above. Proteomic analysis In order to partially digest VDAC1 labeled by PUQ-1 or PUQ-2, VDAC1 was purified by hydroxyapatite chromatography, conjugated with a fluorescent TAMRA–azido tag, separated on a 12.5% Laemmli-type SDS gel, and stained by CBB.

The CBB-stained

VDAC1 band was digested in a 20% Tris-EDTA mapping gel using V8-protease (1 µg/well, 0.5–1 h) or lysylendopeptidase (Lys-C, 1 µg/well, 2 h) according to the procedures described previously (35 and 36).

The partial digests were analyzed by mass

spectrometry or N-terminal sequence analysis.

Regarding exhaustive digestion,

TAMRA-conjugated VDAC1 was digested with Lys-C in buffer containing 50 mM Tris/HCl and 0.1% SDS (pH 8.5).

Digests were separated on a Schägger-type SDS gel

(16.5% T, 6% C containing 6.0 M urea, ref. 37). In the mass spectrometric analysis of proteins, CBB-stained protein bands were in gel digested with trypsin in buffer containing 25 mM NH 4 HCO 3 at 37˚C overnight. 10 ACS Paragon Plus Environment

The

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tryptic digests were desalted with ZipTip (Millipore) and spotted onto the target plate using CHCA as a matrix (27).

A mass spectrometric analysis was conducted using a

Bruker Autoflex III Smartbeam instrument (MALDI-TOF/TOF, Bruker Daltonics).

The

MS spectra obtained were analyzed according to the previous procedures (27). N-terminal amino acid residues were examined with a Procise 494 HT protein sequencing system (Applied Life Sciences, Foster City, CA) at the APRO Life Science Institute, Inc. (Tokushima, Japan). Measurements of mitochondrial swelling and oxygen consumption Mitochondrial swelling and oxygen consumption were examined as reported previously (38).

Briefly, mitochondria (0.15 mg protein/ml) suspended in incubation

medium (0.3 M mannitol, 10 mM HEPES, 0.5 mg/ml BSA, 25 µM EGTA, and 2 mM KPi; pH7.4) were energized with 2 mM NADH in a total volume of 2.2 ml at 25 °C. Mitochondrial swelling was monitored by measuring absorbance at 540 nm with a Shimadzu spectrophotometer, model UV-1700.

Mitochondrial respiration in the same

medium was measured with a Clark-type oxygen electrode in a total volume of 2.2 ml.


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RESULTS Synthesis of photoreactive UQs (PUQ-1 and PUQ-2) Photoaffinity labeling is a powerful chemistry-based means for probing interactions between biologically active chemicals and their target proteins (39).

For example, using

a photoreactive UQ derivative (PUQ-1, Figure 1), we previously demonstrated that mitochondrial Coq10 from Schizosaccharomyces pombe, a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain superfamily, specifically accommodates the quinone-head ring of PUQ-1 in its START domain (27). In the present study, we newly synthesized PUQ-2 (Figure 1), a geometrical isomer of PUQ-1, which possesses a photolabile azido (–N=N + =N - ) group at the 3-positions in the quinone ring.

Concerning PUQ-1 and PUQ-2, two isoprene units were directly

connected to the quinone ring to imitate natural ubiquinones as much as possible.

An

alkyne (–CºCH) was attached to the terminal end of their hydrophobic tails to allow the conjugation of molecular tags, such as fluorophores and biotin (Figure 1), via Cu + catalyzed click chemistry (i.e., azide-alkyne [3+2] cycloaddition in water, ref. 40).

We

did not introduce a photolabile group into the tail moiety of UQ because it is too flexible to fix the position of a photolabile group at the protein-bound state, which may enhance the probability of non-specific labeling.

We herein focused on the specific interaction

between the quinone-head ring and target protein (VDAC). Photoaffinity labeling of yeast mitochondria using PUQ-1 and PUQ-2 Mitochondria (0.6 mg of protein/mL), isolated from S. cerevisiae, were incubated with 2.0 µM PUQ-1, irradiated with a UV lamp, and then covalently conjugated with a fluorescent TAMRA–azido tag (Figure 1) via Cu + -catalyzed click chemistry for visualization of the labeled proteins.

Mitochondrial proteins were separated on a 15%

Laemmli-type SDS gel, followed by CBB staining and fluorescent gel imaging (Figure 2A).

Only a limited number of proteins were strongly labeled by PUQ-1 with a

predominant fluorescent band at ~30 kDa.

When yeast mitochondria were labeled by

PUQ-2, the labeling pattern was almost identical to that by PUQ-1, while the labeling yield of PUQ-2 was considerably lower than that of PUQ-1 (Figure 2B). 12 ACS Paragon Plus Environment

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The mitochondrial proteins labeled by PUQ-1 were then resolved using conventional two-dimensional gel electrophoresis (IEF/SDS-PAGE) (Figure 3A).

We note that a

biotin–azido tag (Figure 1), in place of TAMRA–azido tag, was used to analyze the proteins labeled by PUQ-1 on the 2D gel because the recovery of mitochondrial proteins during the 2D-electrophoretic operation was poor due to their hydrophobic properties. Biotinylated proteins were detected by alkaline phosphatase (AP)-conjugated streptavidin. We detected a number of spots on the gel (Figure 3B).

The signal intensity of the ~30

kDa protein appeared to be weaker than that observed on the 1D gel probably because of the hydrophobic properties of mitochondrial membrane protein(s).

In order to assess

the binding specificity of PUQ-1, we conducted a competition test using an excess amount of 2-N 3 -UQ 2 (Figure 1), which also has a photolabile azido group at the 2-position, but lacks the terminal alkyne group that is a handle to be attached with a biotin–azido tag. Several fluorescent spots disappeared or were significantly diminished in the presence of 2-N 3 -UQ 2 (spots 1–7) (Figure 3C), indicating that the binding of PUQ-1 to these proteins is a specific event. The CBB spots corresponding to the Western blot signals (spots 1–7) were excised from the gel and subjected to MALDI-TOF MS analysis.

The results of the MALDI-

TOF MS analysis indicated that spots 1, 2, and 3 solely contain mitochondrial VDAC1 (Supplemental Table S1), suggesting that the ~30 kDa protein in the 1D gel is VDAC1 (32 kDa).

These findings strongly prompted us to investigate the interaction between

VDAC1 and PUQ-1 in more detail.

The labeling of other proteins is not the aim of this

study and, thus, not discussed further (Spots 4 and 6 were found to be aconitase and Dlactate dehydrogenase, respectively.

Spots 5 and 7 could not be identified because of

contamination of multiple proteins).

Protein spots other than spots 1–7 have not been

identified by mass spectrometry. In the photoaffinity labeling of yeast mitochondria with PUQ-1, we did not detect clear cross-linking against the proteins forming the quinone-binding pocket of mitochondrial respiratory enzymes such as SdhD subunit of succinate-quinone oxidoreductase (complex II) and cytochrome b of quinol-cytochrome c oxidoreductase (complex III).

We cannot exclude the possibility that PUQ-1 actually labeled these 13 ACS Paragon Plus Environment

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proteins; however, the intensity of labeling was too weak to be detected by Western blot signals.

The electron transfer activities of PUQ-1, in terms of V max values, as a substrate

for complexes II and III were considerably lower than those of UQ 1 and UQ 2 (~20–40%). Its poor ability as a respiratory substrate may be a cause of the weak cross-linking. Identification of the ~30 kDa protein labeled by PUQ-1 The above results strongly suggested that the labeled ~30 kDa protein is VDAC1; nevertheless, we cannot rule out the possibility that a major labeled protein (besides VDAC1) at ~30 kDa band in 1D gel (Figure 2) may not be resolved in the 2D gel electrophoresis for some reason; for example, serious aggregation due to its strong hydrophobicity.

Accordingly, the clarification of this point is important.

As reported

previously (32), since hydroxyapatite chromatography is an efficient method to purify VDAC1 and several members of solute carrier family 25 (Slc25) such as AAC, we attempted to purify the labeled protein(s) using this technique. Yeast mitochondria (0.6–1.0 mg of protein/mL) were labeled by 5–10 µM PUQ-1, solubilized with 2% (v/v) Triton X-100, and passed through a hydroxyapatite column, followed by conjugation with a fluorescent TAMRA–azido tag via Cu + -catalyzed click chemistry.

The flow-through fraction of hydroxyapatite chromatography contained a

CBB-stained protein band corresponding to the fluorescent ~30 kDa band (Figure 4), which was identified as VDAC1 by MALDI-TOF MS (42% coverage, Supplemental Table S1).

We also conducted the photoaffinity labeling using mitochondria isolated from

Δpor1 S. cerevisiae lacking VDAC1.

As expected, the fluorescent band at ~30 kDa was

not observed in the crude sample or in the flow-through fraction (Figure 4). To further verify the labeled ~30 kDa protein as VDAC1, the protein labeled by PUQ1 was conjugated with a cleavable biotin–SS–azido tag (Figure 1) via Cu + -catalyzed click chemistry, enriched on immobilized streptavidin, and detected with a commercially available anti-yeast VDAC antibody.

As shown in Figure 5, the biotinylated VDAC1

was captured on streptavidin-agarose and released in Laemmli buffer containing 2.5% mercaptoethanol.

Taken together, our results clearly demonstrated that VDAC1 is

labeled by PUQ-1. 14 ACS Paragon Plus Environment

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We also performed the same experiments using PUQ-2 and confirmed that it also specifically labels VDAC1 (data not shown).

We note that a faint fluorescent band

above the VDAC1 band detected in Figure 2, corresponding to the band with an asterisk in Figures 4 and 5, was identified as AAC by MALDI-TOF MS analysis of its tryptic digests (36% coverage).

However, since the reproducibility of labeling to AAC was

poor in every experiment, this minor labeling of AAC was not examined in further detail. Characterization of the binding manner of PUQ-1 to VDAC1 We conducted a competition test between PUQ-1 and other short-chain UQs (UQ 0 and UQ 2 ) to assess the binding specificity of PUQ-1 to VDAC1.

Xie et al. previously

reported that UQ 0 efficiently suppresses the binding of an antibiotic geldanamycin derivative (17AAG) to human VDAC1 (19).

The photoaffinity labeling of yeast

mitochondria by PUQ-1 (2 µM) was conducted in the presence of an excess amount of UQ 0 or UQ 2 (50~100 molar-fold). labeling (Figure 6A).

Both short-chain UQs significantly suppressed the

In this experiment, we had to use an excess amount of competitors

(UQ 0 and UQ 2 ) to completely suppress the labeling by PUQ-1 because the former and the latter are non-covalently and covalently bound ligands, respectively.

The suppressive

effect by UQ 0 was slightly more pronounced than that by UQ 2 , although the latter is more hydrophobic than the former.

Fontaine et al. reported that UQ 0 and UQ 2 both

significantly inhibit the Ca 2+ -induced PT in rat liver mitochondria and that the inhibitory effect of the former is more efficient than that of the latter (20); however, they did not investigate the target protein of short-chain UQs. We next compared binding affinities against VDAC1 between the oxidized and reduced forms of PUQ-1.

The reduced form of PUQ-1, which was prepared by reduction

with sodium dithionite, was subjected to the photoaffinity labeling under anaerobic conditions (nitrogen gas atmosphere).

The results of photoaffinity labeling (both the

labeling pattern and yield) using the reduced form were similar to those obtained using the oxidized form (Figure 6B), indicating that both forms of PUQ-1 bind to VDAC1 with similar affinities. Previous studies demonstrated that cholesterol binds to mammalian VDAC1 in vivo 15 ACS Paragon Plus Environment

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Page 16 of 45

and in vitro (3, 41), however, the binding position(s) and functional role of bound cholesterol are under debate (42).

We examined the effect of cholesterol on the specific

binding of PUQ-1 (5 µM) to mitochondrial VDAC1.

Even when yeast mitochondria

were incubated with an excess amount of cholesterol (100 molar-fold of PUQ-1), the extent of labeling of VDAC1 was only slightly reduced, by ~10–15% of the control. This result strongly suggests that PUQ-1 and cholesterol do not share a common binding position in VDAC1, as discussed later. Localization of the labeled region in VDAC1 To localize the binding site of PUQ-1 in VDAC1, the protein labeled by PUQ-1 was partially purified using hydroxyapatite chromatography, conjugated with a fluorescent TAMRA-azido tag via click chemistry, and partially digested in the gel with V8-protease or Lys-C.

As shown in Figure 7A, partial digestion with V8-protease reproductively

gave a major fluorescent band at ~8 kDa on a 20% Tris-EDTA mapping gel (fragment A), the N-terminal sequences of which were determined to be H 2 N-F 221 ATRY by Edman degradation.

Additionally, MALDI-TOF MS analysis of the tryptic digests of fragment

A revealed that it contains the internal sequences Gln249–Lys267 (m/z 1958.11).

Based

on the theoretical cleavage sites (Figure 7D), fragment A must be the C-terminal region Phe221–Ala283 (6.7 kDa).

We note that a strong CBB band at ~12 kDa, which lacks a

corresponding fluorescent band, contained the internal sequences Ser2–Arg11 (m/z 1120.56), Gln66–Lys84 (m/z 2061.03), and Ser109–Arg124 (m/z 1800.92). Partial digestion with Lys-C provided relatively large digests at the region of 20~25 kDa.

Edman degradation of fragments B and C (indicated by arrows in Figure 7B)

revealed that their N-terminal sequences are H 2 N-L 62 NDKQ and H 2 N-S 2 PPVY, respectively, suggesting that they were truncated by ~5 kDa at their N- and C-terminus, respectively.

These assignments are supported by the facts that tryptic digests Leu85–

Lys95 (m/z 1202.67) and Ser109–Arg124 (m/z 1800.92) were contained in both fragments B and C, while the digest Gln249–Lys267 (m/z 1958.11) was contained solely in fragment B.

We failed to identify smaller fragments around ~10 kDa because multiple sequences

were detected by Edman degradation in the samples excised from regions corresponding 16 ACS Paragon Plus Environment

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Biochemistry

to the fluorescent spots. On the other hand, the exhaustive digestion of the partially purified VDAC1 by LysC afforded a single final digest, the molecular weight of which appeared to be ~3 kDa on a Tricine gel (Figure 7C).

Along with the results obtained from partial digestion

described above, the ~3 kDa digest should be assigned to the region Leu212–Lys234 (2.6 kDa).

Taking all results together, we conclude that the quinone-head ring of PUQ-1

binds to the C-terminal region Phe221–Lys234.

A summary of peptide mapping for the

digestion of VDAC1 is illustrated in Figure 7D.

In Figure 8, the labeled region Phe221–

Lys234 (in red), which connects the 15th and 16th β-strand sheets and faces the intermembrane space, is indicated in the NMR solution structure of human VDAC1 (3). To pinpoint the amino acid(s) labeled by PUQ-1 via MALDI-TOF MS analysis of the tryptic digests of VDAC1, we purified the labeled VDAC1 through its capture on immobilized streptavidin, as described for Figure 5.

However, we were unable to obtain

a sufficient amount of the labeled protein for the mass spectrometric analysis (i.e. a protein level visible by CBB staining).

As discussed in previous photoaffinity labeling

studies including our own (27, 43, 44), this may be due to the inherently low reaction yield of photoaffinity labeling ( UQ 0 ) may not be responsible for 21 ACS Paragon Plus Environment

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their specific binding to VDAC1.

Page 22 of 45

It is interesting to note that UQ 0 inhibited the Ca 2+ -

induced PT in rat liver mitochondria more efficiently than UQ 2 (20, 21). Different groups recently presented the evidence in support of F o F 1 -ATP synthase as the possible molecular identity of the mitochondrial PTP (52-54); however, there is controversy how the pore is formed and modulated by regulatory factors such as Ca 2+ , Pi, and cyclosporin A.

Since F o F 1 -ATP synthase has attracted a lot of attention, many other

protein candidates of the PTP (such as ANT, PiC, and VDAC), which have long been investigated, seem to become the subject of intense debate (11).

However, more recent

RNAi-based studies using human cells revealed an important regulatory role of VDAC in the mitochondrial PTP opening in combination with other mitochondrial proteins (15, 16). In the present study, we demonstrated that PUQ-1 and PUQ-2 significantly inhibit the Ca 2+ -induced yeast mitochondrial swelling, which is one of the typical changes accompanying the induction of mitochondrial PT (20).

Taking their binding

specificities against VDAC1 into consideration, these inhibitory effects may be attributable to the direct interaction between quinones and VDAC1.

The features of

yeast mitochondrial PT are distinct from those of mammalian mitochondrial PT in several aspects; for example, yeast PT is not inhibited by cyclosporin A (38, 55).

Further studies

are necessary in order to clarify how quinone bound to VDAC1 modulates its regulatory role in mitochondrial PT. The biosynthesis of natural UQ is completed in the mitochondrial inner membrane of eukaryotes (56), and ubiquinone can migrate to the mitochondrial outer membrane through hitherto unknown processes (57).

The possibility of UQ biosynthesis in the

extra-mitochondrial space has also been suggested (47).

Thus, natural ubiquinone

comes into contact with VDAC1 in the outer membrane under physiological conditions. The present study did not directly prove that native ubiquinone (UQ 6 in S. cerevisiae) binds to VDAC1 and modulates its function in the regulation of mitochondrial PT; hence, it is currently unclear whether natural ubiquinone behaves in a similar manner to our photoreactive quinones.

Nevertheless, the results of the present study would provide a

basis for elucidating the regulatory role of ubiquinone in mitochondrial PT.


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Biochemistry

ACKNOWLEDGEMENTS We thank Drs. Fumihiko Sato and Kentaro Ifuku, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, for allowing us access to their MALDITOF MS (Bruker Autoflex III Smartbeam) as well as for their helpful advice on the experiments. AUTHOR INFORMATION Corresponding author: *Hideto Miyoshi, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. E-mail: [email protected]. Tel: +81-75-753-6119 Funding source information: This study was supported by a Grant-in-Aid for Scientific Research (Grant 26292060 to H. M. and Grant 15K07411 to M. M.) from the Japan Society for the Promotion of Science.

SUPPORTING INFORMATION AVAILABLE Synthesis of PUQ-2. organisms.

Figure S1: Multiple sequence alignment of VDAC1 from various

Table S1: MALDI-TOF MS analysis of the tryptic digests of VDAC1.

Table S2: List of the primers prepared in this study.

This material is available free of

charge via the Internet at http://pubs.acs.org.


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Page 24 of 45

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40. Wang, Q., Chan, T.R., Hilgraf, R., Fokin, V. V., Sharpless, K. B., and Finn, M. G. (2003) Bioconjugation by copper (I)-catalyzed azide-alkyne [3+2] cycloaddition. J. Am. Chem. Soc. 125, 3192-3193 41. De Pinto, V., Benz, R., and Palmier, F. (1989) Interaction of non-classical detergents with the mitochondrial porin, a new purification procedure and characterization of the pore-forming unit. Eur. J. Biochem. 183, 179-187 42. Weiser, B. P., Salari, R., Eckenhoff, R. G., and Brannigan, C. (2014) Computational investigation of cholesterol binding site on mitochondrial VDAC. J. Phys. Chem. B 118, 9852-9860 43. Shinohara, H., Ogawa, M., Sakagami, Y., and Matsubayashi, Y. (2007) Identification of the ligand binding site of phytosulfokine receptor by on-column photoaffinity labeling. J. Biol. Chem. 282, 124-131 44. Murai, M., Yamashita, T., Senoh, M., Mashimo, Y., Kataoka, M., Kosaka, H., Matsuno-Yagi, A., Yagi, T., and Miyoshi, H. (2010) Characterization of the ubiquinone

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FIGURE LEGENDS Figure 1. Structures of PUQ-1, PUQ-2, 2-N 3 -UQ 2 , and chemical tags (fluorescent TAMRA–azido and biotin–SS–azido) used in this study. Figure 2. Photoaffinity labeling of yeast mitochondria by PUQ-1 or PUQ-2. (A) Yeast mitochondria (0.6 mg of protein/mL) were labeled with 2 µM PUQ-1 in the presence of 50 µM NADH and denatured with 1% (w/v) SDS, followed by conjugation with a fluorescent TAMRA–azido tag via Cu + -catalyzed click chemistry.

Proteins were

separated on a 15% Laemmli-type SDS gel, and subjected to fluorescent gel imaging and CBB staining.

Control 1; without PUQ-1, control 2; with 2 µM PUQ-1 but without

TAMRA–azido tag; control 3; without PUQ-1 but with TAMRA–azido tag.

(B) The

efficiency of photoaffinity labeling was compared between PUQ-1 and PUQ-2 under the same experimental conditions as those in (A).

Approximately 40 µg of mitochondrial

proteins was loaded in each well. Figure 3. Resolution of yeast mitochondrial proteins labeled by PUQ-1 on two-dimensional gels. Yeast mitochondria (0.6 mg of protein/mL) were labeled with 5 µM PUQ-1 in the presence of 50 µM NADH, followed by conjugation with a biotin–azido tag via Cu + catalyzed click chemistry.

Proteins were separated by the first dimensional IEF (pH 3–

10) and by second dimensional 15% Laemmli-type SDS-PAGE.

The resolved proteins

were stained by CBB or, alternatively, transferred onto a PVDF membrane followed by colorimetric detection using streptavidin-AP (alkaline-phosphatase).

In order to assess

the binding specificity of PUQ-1, a competition test was conducted using an excess amount of 2-N 3 -UQ 2 (100 µM).

The train of spots marked by black arrowheads (spots

1-3) was identified as VDAC1 by MALDI-TOF MS.

Other candidates for “ubiquinone-

binding proteins” are indicated as white arrowheads.


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Biochemistry

Figure 4. Partial purification of VDAC1 using hydroxyapatite chromatography. Yeast mitochondria isolated from wild-type and Δpor1 mutant S. cerevisiae cells (0.6 mg of protein/mL) were labeled with 2 µM PUQ-1 and partially purified by hydroxyapatite chromatography, followed by conjugation with a fluorescent TAMRA–azido tag. Proteins (input and elute) were separated on a 15% Laemmli-type SDS gel and subjected to CBB staining and fluorescent gel imaging. coverage).

Arrowheads indicate VDAC1 (42%

The ~34 kDa protein marked with an asterisk was identified as an ADP/ATP

carrier (36% coverage by MALDI-TOF MS). Figure 5. Specific capture of VDAC1 labeled by PUQ-1 using immobilized avidin. Yeast mitochondria (0.8 mg of protein/mL, 200 µL) were labeled with 10 µM PUQ-1 and conjugated with a cleavable biotin–SS–azido tag via click chemistry.

Biotinylated

proteins were enriched using streptavidin-agarose, according to the procedure described in the Experimental procedures.

Mitochondria treated without PUQ-1 were used as a

negative control (Cont. vs. PUQ-1).

The enriched proteins were released from

streptavidin in Laemmli’s buffer containing 2.5% mercaptoethanol at 40˚C for 1 h (elute). The specific capture and release steps were monitored by a Western blot analysis using an antibody against yeast VDAC (A) or by sliver stain (B). indicated by an arrowhead.

The position of VDAC1 is

For the analysis of total proteins (input) and unbound

fraction (unbound), approximately 6 µg of proteins were loaded on each well. Figure 6. Specificity of the photoaffinity labeling of VDAC1 by PUQ-1. (A) Competition test between PUQ-1 and UQ 0 or UQ 2 .

Yeast mitochondria (0.6 mg of

protein/mL, 200 µL) were labeled with 2 µM PUQ-1 in the presence of UQ 0 (100 µM) or UQ 2 (100 and 200 µM).

The labeled proteins were conjugated with a fluorescent

TAMRA–azido tag via click chemistry, followed by resolution on a 15% Laemmli-type SDS gel.

(B) Comparison of the binding affinities between the oxidized and reduced 31 ACS Paragon Plus Environment

Biochemistry

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forms of PUQ-1.

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Yeast mitochondria (0.6 mg of protein/mL, 200 µL) were labeled with

the oxidized or reduced forms of PUQ-1 (5 µM each), followed by the conjugation with a fluorescent TAMRA tag.

Approximately 40 µg of mitochondrial proteins were loaded

on each well. Figure 7. Localization of the region labeled by PUQ-1 in VDAC1. Yeast mitochondria (0.6 mg of protein/mL, 1.0 mL) were labeled with 5 µM PUQ-1, and VDAC1 was partially purified using hydroxyapatite chromatography, followed by conjugation with a fluorescent TAMRA–azido tag. separated on a 15% Laemmli-type SDS gel.

(A) TAMRA-attached VDAC1 was

A CBB-stained gel piece of VDAC1 was

subjected to in gel partial digestion with V8-protease according to the procedure described in the Experimental Procedures section.

The N-terminal sequences

corresponding to the fluorescent band (Fragment A) were determined by Edman degradation.

(B) The partial digestion of VDAC1 was also performed using Lys-C to

provide Fragments B and C.

(C) Purified VDAC was exhaustively digested with Lys-C,

followed by separation on a 16.5% Schägger-type SDS gel. of the partial and exhaustive digestion of VDAC1.

(D) Schematic presentation

The predicted cleavage sites are

denoted with arrows and indicated by their residue numbers in the mature sequences of S. cerevisiae VDAC1 (SwissProt entry P04840). Figure 8. PUQ-1 (PUQ-2)-binding site in VDAC1 of S. cerevisiae mitochondria. The structure of human VDAC1 (Protein data bank entry 2K4T) revealed by NMR study from side (A) and top (B) views (rotated by 90˚).

Using a multiple sequence alignment

for S. cerevisiae and human VDAC1 (Supplemental Figure S1), the sequence corresponding to the region labeled by PUQ-1 (Phe221–Lys234) is colored in red. Amino acids mutated in this study are shown as stick models. Figure 9. 32 ACS Paragon Plus Environment

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Biochemistry

Photoaffinity labeling using yeast VDAC1 mutants. Yeast mitochondria expressing wild-type or mutant VDAC1 (0.6 mg of protein/mL, 200 µL) were labeled with 2 µM PUQ-1 and conjugated with a fluorescent TAMRA–azido tag, followed by resolution on a 15% Laemmli SDS gel.

The extent of the incorporation of

PUQ-1 into the VDAC1 mutants and the expression levels of these mutants were assessed by fluorescent intensities (upper) and a Western blot analysis using an anti-yeast VDAC antibody (lower), respectively.

Approximately 40 and 20 µg of proteins were loaded in

each well for fluorescent gel imaging and Western blot analysis, respectively. representative

of

three

independent

experiments

using

separate

Data are

mitochondrial

preparations. Figure 10. Effects of UQ analogues on the Ca 2+ -induced mitochondrial PT. Yeast mitochondria (0.15 mg of protein/mL) were suspended in incubation medium described in the Experimental Procedures section and energized with 2 mM NADH. Mitochondrial PT was induced by adding 100 µM Ca 2+ , and swelling was monitored by following absorbance changes at 540 nm.

The traces obtained are as follows: trace 1,

control; trace 2, 5 µM UQ 0 ; trace 3, 5 µM PUQ-1; trace 4, 5 µM PUQ-2; trace 5, 10 mM KPi.

Data are representative of three independent experiments using separate

mitochondrial preparations. Figure 11. Effects of UQ analogues on mitochondrial respiration rates. Yeast mitochondria (0.15 mg of protein/mL) were suspended in incubation medium described in Figure 10, and NADH-energized respiration was monitored with a Clarktype oxygen electrode.

The traces obtained are as follows: trace 1, in the absence of

exogenous UQ (a control); trace 2, 5.0 µM UQ 0 ; trace 3, 5.0 µM PUQ-1; trace 4, 5.0 µM PUQ-2; trace 5, 0.2 µM antimycin A.

Data are representative of three independent

measurements using separate mitochondrial preparations.


Biochemistry

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O O

3

N3

2

PUQ-1

5

O

6

O

O

2

4

O N3

PUQ-2

O

O O

O

2

4

O O

2-N3-UQ2

N3

O N O

TAMRA-azido tag HOOC H N

N3

N O O

biotin-azido tag O N3

N

4 H

biotin-SS-azido tag H N S N3 4

O

HN H

H N 4

S O

S

O HN H

H N

O

NH H

NH H S

O

Figure 1


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Biochemistry

Figure 2


Biochemistry

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Figure 3


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Biochemistry

Figure 4


Biochemistry

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Figure 5


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Biochemistry

Figure 6


Biochemistry

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Figure 7


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Biochemistry

Figure 8


Biochemistry

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Figure 9


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Biochemistry

Figure 10


Biochemistry

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Figure 11


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Biochemistry

FOR TABLE OF CONTENTS ONLY

Synthetic Ubiquinones Specifically Bind to Mitochondrial Voltage-Dependent Anion Channel 1 (VDAC1) in Saccharomyces cerevisiae Mitochondria Masatoshi Murai 1 , Ayaka Okuda 1 , Takenori Yamamoto 2 , Yasuo Shinohara 2 , and Hideto Miyoshi 1* 1

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,

Sakyo-ku, Kyoto 606-8502, Japan, and 2 Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan.


Synthetic Ubiquinones Specifically Bind to Mitochondrial Voltage

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