Changes in specific lipids regulate BAX-induced mitochondrial permeability transition

Changes in specific lipids regulate BAX-inducedmitochondrial permeability transitionE. Martınez-Abundis1, N. Garcıa1, F. Correa1, M. Franco2 and C. Zazueta1

1 Departamento de Bioquımica, Instituto Nacional de Cardiologıa Ignacio Chavez, Mexico

2 Departamento de Nefrologıa, Instituto Nacional de Cardiologıa Ignacio Chavez, Mexico

BAX is a pro-apoptotic member of the Bcl-2 protein

family that resides in an inactive state in the cytoplasm

of normal cells. Following an apoptotic stimulus, BAX

undergoes conformational changes [1–4], becoming a

mediator of the intrinsic phase of apoptosis by its

insertion into mitochondrial membranes, a process that

culminates in the release of cytochrome c and activa-

tion of effector caspases [5]. Evidence from the

literature indicates that BAX is activated by other

BH3-only proteins, particularly Bid protein and Bim

peptide [6,7]. In this respect, it has been shown that

Bid activation depends on its proteolytic processing

into tBid and the translocation of tBid to mitochon-

dria [8]. In the mitochondria, tBid may form oligomers

by itself and induce oligomerization of BAX and Bak

[9]. Although it remains unclear how tBid triggers such

oligomerization, there is strong evidence to suggest

that Bid-induced apoptosis depends on the presence of

BAX and Bak [10]. However, it has also been demon-

strated that tBid accumulation in mitochondria is an

essential, but not sufficient, event leading to mito-

chondrial disruption for cytochrome c leakage [6]. The

Keywords

BAX; cholesterol; gangliosides; lipid

microdomains; mitochondrial permeability

transition pore

Correspondence

C. Zazueta, Instituto Nacional de Cardiologıa,

I. Ch., Departamento de Bioquımica, Juan

Badiano No. 1, Colonia Seccion XVI,

Mexico 14080, D.F

Fax: +52 55 5573 0926

Tel: +52 55 5573 2911 (1465)

E-mail: [email protected]

(Received 13 August 2007, revised 17

October 2007, accepted 25 October 2007)

doi:10.1111/j.1742-4658.2007.06166.x

Recent evidence suggests the existence of lipid microdomains in mitochon-

dria, apparently coexisting as structural elements with some of the mito-

chondrial permeability transition pore-forming proteins and members of

the Bcl-2 family. The aim of this study was to investigate the relevance of

the main components of membrane microdomains (e.g. cholesterol and

sphingolipids) in activation of the mitochondrial permeability transition

pore (mPTP) by recombinant BAX (rBAX). For this purpose, we used

chemically modified renal cortex mitochondria and renal cortex mitochon-

dria from hypothyroid rats that show a modified mitochondrial lipid com-

position in vivo. Oligomeric rBAX induced an enhanced permeability

conformation in the mPTP of control mitochondria. rBAX failed to induce

mPTP opening when the cholesterol and ganglioside content of mitochon-

dria were modified with the chelator methyl-beta-cyclodextrin. Accordingly,

hypothyroid mitochondria, with endogenously lower cholesterol and gan-

glioside content, showed resistance to mPTP opening induced by rBAX.

These observations suggest that enriched cholesterol and ganglioside

domains in the mitochondrial membranes may determine BAX interaction

with the mPTP. An intriguing observation was that chemical extraction of

cholesterol and ganglioside in control mitochondria did not have an effect

on rBAX insertion. Conversely, in hypothyroid mitochondria, rBAX inser-

tion was diminished dramatically compared with control mitochondria.

The membrane and protein changes associated with thyroid status and

their possible role in rBAX docking into the membranes are discussed.

Abbreviations

ANT, adenine nucleotide translocator; CSA, cyclosporine A; GST, glutathione S-transferase; MbCD, methyl-beta-cyclodextrin; mPT,

mitochondrial permeability transition; mPTP, mitochondrial permeability transition pore; rBAX, recombinant BAX; VDAC, voltage-dependent

anion channel; Dw, transmembrane potential.

6500 FEBS Journal 274 (2007) 6500–6510 ª 2007 The Authors Journal compilation ª 2007 FEBS

molecular mechanism underlying such leakage is still a

matter of debate. One proposal is that oligomeric

BAX forms pores in the outer mitochondrial mem-

brane, providing a mechanism for cytochrome c release

[11]; a second proposal is that BAX induces opening

of the permeability transition pore (mPTP) [12]. The

opening of this mega-channel would lead to mitochon-

drial swelling and rupture of the outer membrane, thus

explaining the release of cytochrome c from mitochon-

dria. Although several reports have suggested an inter-

action between BAX and the components of the

mPTP, e.g. the adenine nucleotide translocator (ANT)

[13] and ⁄or the voltage-dependent anion channel

(VDAC) [14], the factors involved in BAX induction

of mitochondrial permeability transition (mPT) are

unknown.

By contrast, lipid microdomains or ‘lipid rafts’ were

first described in the plasma membrane. It is known

that they are composed mainly of sphingolipids and

cholesterol, and have been considered to be architec-

tural domains, where specific proteins could interact to

exert specific regulatory mechanisms [15].

Relevant to this issue, there is recent evidence sug-

gesting the existence of lipid microdomains in mito-

chondria which coexist as structural elements with

some of the mPTP-forming proteins [16]. Recent

reports indicate that the disialoganglioside (GD3) con-

tributes directly to the opening of the permeability

transition pore complex in isolated mitochondria

[17,18] and furthermore, that GD3-induced damage in

mitochondrial membranes is confined to sites that can

be restrained by Bcl-2 [19]. Also, several lines of evi-

dence converge on the assumption that the mPTP prop-

agates the downstream stage of apoptosis, mediated by

the lipid-dependent pathway, which includes ceramide

and GD3 [20]. In addition, cholesterol is known to

induce changes in the phospholipid packing of the lipid

bilayer and has been suggested as critical for micro-

domain formation [21]. Finally, Keller et al. [22]

reported that changing the nature of the sterol in cellu-

lar rafts leads to changes in the protein composition of

those domains. Our results suggest that cholesterol and

GD3 are relevant for BAX interaction with the mPTP.

These data provide evidence that specific lipids play a

key role in cross-talk between rBAX and the mPTP.

Results

rBAX-induced mitochondrial calcium release and

modified cytochrome content

The addition of oligomeric rBAX to isolated mito-

chondria induced the release of accumulated calcium

in a dose-dependent manner (Fig. 1A). Figure 1 also

shows that 1 lm carboxyatractyloside, a well-known

mPTP inducer, promoted immediate depletion of the

intramitochondrial calcium content. The recombinant

protein associated with mitochondrial membranes was

resistant to alkaline extraction, indicating that it was

deeply embedded in the bilayer (Fig. 1B). A clear cor-

respondence between cytochrome c release and calcium

extrusion from the mitochondria was observed by

inducing opening of the mPTP with either carboxya-

tractyloside or rBAX (Fig. 1C).

Cyclosporin A prevented cytochrome c release

and mPTP opening induced by rBAX

The immunosuppressant cyclosporin A (CSA) effec-

tively inhibits mPTP opening under almost all condi-

tions, and is therefore considered a marker for mPT.

To determine whether the altered mitochondrial per-

meability observed in the presence of rBAX in rat

kidney mitochondria was related to the opening of

this mega-channel, we measured the effect of CSA

on calcium release induced by the addition of recom-

binant protein (Fig. 2A). CSA inhibited calcium

release, although rBAX remained attached to the

membranes (Fig. 2B). Interestingly, mitochondria

incubated with rBAX only released cytochrome c

when the permeability transition pore was opened,

and this was abolished in a medium supplemented

with CSA (Fig. 2C). These results suggest active par-

ticipation of the mPTP on cytochrome c release

induced by rBAX.

The effect of rBAX on the mPT was entirely

dependent on calcium. Typical traces of mitochon-

drial swelling with and without calcium are shown in

Fig. 3A. An important decrease in light scattering

was detected when the medium was supplemented

with a final calcium concentration of 50 lm, and was

inhibited completely by 1 lm CSA. Also, pore open-

ing can be estimated by following the discharge

kinetics of the transmembrane potential (Dw). Fig-

ure 3B shows that the membrane potential developed

by mitochondria added to the medium without any

previous treatment (trace a) was similar to that devel-

oped in mitochondria incubated with rBAX in the

absence of calcium (trace b). Conversely, addition of

50 lm CaCl2 to the medium led to a decrease in the

Dw of mitochondria incubated with rBAX, which

eventually resulted in its total collapse (trace c). We

also determined mitochondrial NAD+ content, which

has been related to mPTP gating in situ. The NAD+

content of mitochondria incubated for 15 min with

rBAX, but not exposed to the calcium-containing

E. Martınez-Abundis et al. Microdomain components – effect on BAX-induced mPT

FEBS Journal 274 (2007) 6500–6510 ª 2007 The Authors Journal compilation ª 2007 FEBS 6501

medium, was similar to control mitochondria that

had undergone incubation and further exposure to a

medium supplemented with calcium, but not incu-

bated with rBAX (9.66 versus 9.38 nmol NAD+Æmg

protein)1, n ¼ 2). Conversely, NAD+ content

decreased in rBAX-treated mitochondria when mPTP

was induced in a medium supplemented with calcium

(2.08 nmol NAD+Æmg protein)1, n ¼ 2).

Lipid modification in rat kidney mitochondria

produced resistance to rBAX-induced

permeability transition

Methyl-beta-cyclodextrin (MbCD) is known to induce

cholesterol efflux from membranes and, consequently,

to promote microdomain disruption. We evaluated the

effect of this chelator on the ability of rBAX to induce

M

Time Course Graph

750

Control,

Cal

cium

rel

ease

rBAX + CSA

0 1500Time (seconds)

0.35Mitos ± rBAX

A 0.18

0.02

rBAX

BAX

Cytc

A B

Con

trol

rBA

X

rBA

X +

CS

A

Std

SS

M

Fig. 2. CSA prevents cytochrome c release

and mPTP opening induced by rBAX. (A)

After treatment of mitochondria with

150 nM rBAX, mPTP opening was evaluated

as calcium release under the conditions

described in Fig. 1. Where indicated, the

assay medium was supplemented with

1 lM CSA. (B) rBAX and cytochrome c con-

tent was detected in mitochondria, as

described in Fig. 1. Traces and blots are rep-

resentative of at least four different experi-

ments. M, mitochondria; SS, supernatants;

Std, purified rBAX and commercial cyto-

chrome c, respectively.

M

SS

CAT 1 μM

(nM)

200

100

50

M± rBAX

Time course graph

0 1090 2200 Time (seconds)

0.30

0.16

0.02

Cal

cium

rele

ase

rBAX (nM)

200 100 - 1

CAT (μM)

ANT

200

rBAX (nM)

BAX

A B

Cytc M

A

100 50 -

Fig. 1. Recombinant BAX induces mitochondrial calcium extrusion and cytochrome c release from isolated mitochondria. (A) Calcium uptake

in isolated mitochondria in the presence of different rBAX concentrations. Mitochondrial protein (1.3 mg) was incubated with oligomerized

rBAX, as described in Experimental procedures, for 15 min and then added to 2 mL of a medium containing 125 mM KCl, 10 mM Tris,

10 mM succinate, 3 mM Pi, 200 lM ADP, 50 lM CaCl2, 3 lgÆmL)1 rotenone and 50 lM Arsenazo III, pH 7.4. (B) BAX detection in mitochon-

dria. After maximal calcium release, 1.8 mL of the suspension was withdrawn and centrifuged at 18 000 g for 10 min. Mitochondrial sam-

ples were treated with Na2CO3 as described in Experimental procedures. Mitochondria were dissolved in 50 lL NaCl ⁄ Pi, pH 7.0, and mixed

with 25 lL of a 3· Laemmli’s loading buffer, boiled for 15 min, subjected to SDS ⁄ PAGE and evaluated by western blot. Protein loading was

determined by using anti-ANT polyclonal IgG. (C) Cytochrome c content in mitochondria (M) and in supernatants (SS) recovered after rBAX

incubation. Proteins in the supernatants were precipitated with trichloroacetic acid and evaluated for cytochrome c content along with mito-

chondrial samples. Results are representative of three independent experiments.

Microdomain components – effect on BAX-induced mPT E. Martınez-Abundis et al.


opening of the permeability transition pore in rat

kidney mitochondria. MbCD at doses of 0.5–1.5 mm

prevented opening of the mPTP induced by 150 nm

oligomeric rBAX (Fig. 4A). Interestingly, rBAX

remained inserted into MbCD-treated mitochondria

(Fig. 4B). These results indicate that altered cholesterol

levels in mitochondrial membranes are important for

rBAX cross-talk with components of the mPTP, and

do not influence rBAX insertion.

Kidney mitochondria from hypothyroid rats were

refractive to mPTP-opening induced by rBAX

To gain insight into the existence of putative mito-

chondrial lipid microdomains in vivo, we evaluated the

inducing effect of rBAX on opening of the mPTP

in kidney mitochondria from hypothyroid rats. We

hypothesized that if cholesterol and gangliosides are

required for BAX-induced mPTP opening, and if such

0 500 1000Time (seconds)

Mem

bran

e po

tent

ial

(–)

(+)

Time course graph

0.30

0.25

0.20

0.15

0.10

A

d

a

b

c

A B


0.80

0.70

0.60

a, bcd

e

M±rBAX

Time course graph

Mito

chon

dria

l sw

ellin

g

A

Fig. 3. Effect of rBAX on mitochondrial swelling and Dw. (A) Mitochondrial swelling induced by rBAX was monitored as absorbance changes

at 540 nm. Mitochondrial protein (1.3 mg) was incubated with 150 nM oligomerized rBAX and added to 2 mL of basic medium, described in

Experimental procedures, without calcium. Where indicated, the medium was supplemented with 50 lM CaCl2 and 1 lM CSA. Trace a, mito-

chondria incubated only in the presence of the octyl glucoside (OG) concentration that promotes rBAX oligomerization; trace b, as in trace a,

but the medium contained CaCl2; trace c, mitochondria incubated with 150 nM rBAX in the presence of calcium and CSA; trace d, mitochon-

dria incubated with 150 nM rBAX without calcium; trace e, mitochondria incubated with 150 nM rBAX and calcium. (B) Dw in rBAX-treated

mitochondria. Mitochondria added to the calcium-containing medium without any previous treatment (trace a). Mitochondria incubated with

rBAX without calcium (trace b). Mitochondria incubated with rBAX and calcium (trace c).

A

a

b, c

rBAX

BAX

MβCD – – +

A

B

VDAC

0

0.04

0.06

0.08

0.10

0.12


Time course graph

Cal

cium

rel

ease

Fig. 4. MbCD inhibits mPTP opening induced by rBAX in control mitochondria. (A) Oligomerized rBAX was incubated with control mitochondria

for 15 min and then added to the basic medium described in Fig. 1, in the presence of MbCD. Trace a, 150 nM rBAX; trace b, 150 nM rBAX and

0.5 mM MbCD; trace c, 150 nM rBAX and 1.5 mM MbCD. [Correction added after publication 26 November 2007: Fig. 4A has been replaced with

a corrected version] (B) Mitochondrial samples were withdrawn at the end of the tracing, centrifuged and subjected to alkaline extraction before

SDS ⁄ PAGE fractioning and western blotting. The horizontal line indicates the samples with added rBAX. Membranes were incubated against

anti-BAX mAb, stripped, and evaluated for VDAC content to check protein loading. Blots are representative of two different experiments.



domains could be key sites for BAX docking and

anchoring to the membrane, we should be able to find

a different response from either hypothyroid or control

mitochondria, on account of their modified mitochon-

drial lipid composition [23]. Figure 5A shows that

150 nm rBAX did not have any effect on mPTP open-

ing in hypothyroid mitochondria; furthermore, rBAX

insertion was dramatically diminished in mitochondrial

membranes (Fig. 5B).

Comparison between cholesterol and ganglioside

content in chemically modified rat kidney mito-

chondria and hypothyroid kidney mitochondria

The cholesterol content of control and hypothyroid

mitochondria was measured using gas chromatogra-

phy. In hypothyroid kidney mitochondria, cholesterol

content was 39% lower than in control (euthyroid)

mitochondria, i.e. 43.1 ± 1.6 versus 70.5 ± 14.7

lgÆmg)1 protein). Diminished cholesterol levels in

mitochondria correlated with inhibition of the ‘open

state’ of the mPTP induced by rBAX. Interestingly,

chemical cholesterol depletion of control mitochondria

membranes by MbCD (34%) produced resistance to

rBAX-induced permeability transition (Table 1). These

results suggest that specific lipid domains are required

for BAX interaction with mPTP components; alterna-

tively, cholesterol may be important for correct assem-

bly of the pore components after rBAX induction of

the ‘open state’.

Along with cholesterol, glycosphingolipids are

concentrated in specific lipid domains in the plasma

membrane. Recently, evidence has indicated that gly-

cosphingolipids and their precursor, ceramide, are also

associated with intracellular organelles, particularly

mitochondria [24]. Because GD3 is the main glyco-

sphingolipid associated with mitochondria and it has

been shown to have a role in mPTP regulation, we

analyzed the polar glycosphingolipid fraction from

total mitochondrial lipid extracts. The obtained glycos-

phingolipids were dried and separated by TLC. Only

one positive band was found after sulfuric acid detec-

tion that comigrated with the GD3 standard in control

mitochondria (not shown). Indeed, GD3 in hypothy-

roid mitochondria was almost undetectable after TLC.

To verify that GD3 signal variation was not due to

variable loading, we performed densitometric scanning

analysis of plates in which equal volumes of extracted

Control Hypothyroid

M BAX

VDAC

rBAX - - - + + + +

A B

A

0

0.10

0.15

0.20

0.05


Cal

cium

rel

ease

a

b

cd

M±rBAX

Time course graph

Fig. 5. Hypothyroid mitochondria are resistant to mPT induced by rBAX. (A) Calcium release was measured in control and hypothyroid rat

kidney mitochondria as described in Fig. 1. Trace a, control mitochondria incubated with 150 nM rBAX; trace b, control mitochondria incu-

bated with medium without rBAX; trace c, hypothyroid mitochondria incubated in the presence of 150 nM rBAX; trace d, hypothyroid mito-

chondria incubated without rBAX. Results are representative of at least three independent experiments in which different mitochondria and

rBAX preparations were used. (B) Mitochondrial samples were withdrawn at the end of the tracing, centrifuged and subjected to alkaline

extraction before SDS ⁄ PAGE fractioning and western blotting. Membranes were incubated against anti-BAX mAb, stripped, and evaluated

for VDAC content to check loading. Blots are representative of three different experiments. M, mitochondria.

Table 1. Cholesterol content in control and hypothyroid mitochon-

dria after MbCD treatment. Mitochondria from control and hypothy-

roid rats were incubated with 1.5 mM MbCD, as indicated

previously. Total lipids were recovered and evaluated for choles-

terol content as indicated in Experimental procedures. Data repre-

sent mean ± SD of the indicated number of independent

experiments.

Cholesterol (lgÆmg protein)1)

Control

Hypothyroid

(n ¼ 5)

Dimethylsulfoxide-treated

mitochondria

70.55 ± 14.7 (n ¼ 5) 43.1 ± 1.6

MbCD-treated mitochondria 46.54 ± 17.6* (n ¼ 5) 37.6 ± 1.9*

*P < 0.05 versus mitochondria control.



glycosphingolipid fractions, obtained from the same

amount of mitochondrial protein, were loaded and

developed with sulfuric acid (unspecific) and resorcinol

(ganglioside-specific stain). By increasing the volumes

applied to the plates we detected resorcinol-positive

signaling (Fig. 6A). The ratio between the signals is

plotted in Fig. 6B.

The lower GD3 content observed in hypothyroid

mitochondria is consistent with reports that hypothy-

roid conditions affect the biosynthesis and expression

of gangliosides in specific tissues and cell types [23].

This, along with lower cholesterol levels in the mem-

branes, may translate into a diminished ability to form

lipid microdomains in which specific proteins could be

recruited and possibly, to interact.

Because cardiolipin is relevant for mPTP regulation

and this inner membrane phospholipid is diminished in

thyroid insufficiency, a condition that favors the closed

mPTP conformation, we sought to determine the effect

of MbCD treatment on cardiolipin content in control

and hypothyroid mitochondria. Figure 7A, shows the

chromatogram of the phospholipids extracted from

both mitochondrial types after MbCD incubation. As

expected, cardiolipin content in hypothyroid mitochon-

dria was significantly diminished compared with con-

trol mitochondria, but no changes were observed in

MbCD-treated mitochondria. We conclude that

changes in cardiolipin content could not account for

MbCD protection from mPTP opening, at least in

control mitochondria, but may be important for rBAX

insertion into the mitochondrial membranes.

Discussion

The purpose of this study was to demonstrate the

participation of lipids, specifically those related to

microdomain components, in the activation of the per-

meability transition pore by the pro-apoptogenic pro-

tein BAX, using lipid-modified mitochondria in vitro

and in vivo.

It is widely known that membrane lipids have major

structural and functional roles modulating signaling

pathways. In the plasma membrane, apoptotic death

mediated by lipids is propagated by activation of the

surface receptor CD95 (FAS ⁄APO-1), and includes the

formation of ceramide and GD3 [24]. Ceramide is pro-

duced by the hydrolysis of membrane sphingomyelin

[25] and by means of a GD3 synthase (a2,8-sialyltrans-ferase), rapidly converted to gangliosides [26]. GD3

and cholesterol are relatively enriched in plasma mem-

branes and are concentrated in specialized domains

called rafts or lipid microdomains [15]. In this sense,

new evidence suggests that raft domains are not exclu-

sive to the plasma membrane [16,27]. In particular, the

existence of GD3-enriched microdomains in mito-

chondria [16], and furthermore, data suggesting the

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

A

B

*

**

Hypothyroid+ MβCD

Hypothyroid Control+ MβCD

Control

Sig

nal r

atio

(re

sorc

inol

/H2S

O4)

Control Hypo GD3

Resorcinol

H2SO4

MβCD - - + + - - + +

n= 9 n= 6 n= 5 n= 6

Fig. 6. GD3 content in control and hypothyroid mitochondria after

MbCD treatment. Mitochondrial ganglioside content in which 5 lg

standard GD3 was run in parallel with mitochondrial extracted lipids.

(A) The plate is representative of three independent experiments.

(B) The densitometric ratio between sulfuric acid detection for total

lipids and ganglioside-specific detection with resorcinol is pre-

sented. A value of P < 0.05 was considered statistically significant.

1.5

1

0.5

19.1 ± 5.2*

17.8 ± 6.0*

19.4 ± 2.0*

15.3 ± 4.3*

31.1 ± 6.4

30.1 ± 1.9

25.9 ± 6.4

29.2 ± 5.20

Hypo

(µg CL/mgprotein)

Control

(µg CL/mg protein)

MβCD

(mM)

MβCD (mM)

CL

A

B

0 0.5 1.0 1.5 0 0.5 1.0 1.5 CL

Control Hypo

Fig. 7. Mitochondrial cardiolipin analysis after MbCD-treatment. (A)

Cardiolipin was separated by TLC as described in the Experimental

procedures from control and hypothyroid mitochondria treated with

MbCD. (B) Quantification was performed using acridine orange. Val-

ues are expressed as the mean ± SD of three different experi-

ments. CL, cardiolipin. *P < 0.005 versus respective control.



existence of a metabolic pathway of sphingolipids,

including several enzyme activities of sphingolipid

metabolism, have been reported in these organelles

[28]. Thus, we measured the oligomeric rBAX-inducing

effect on the mitochondrial permeability transition in

chemically modified renal cortex mitochondria and

renal cortex mitochondria from hypothyroid rats,

which show an in vivo modified mitochondrial lipid

composition. According to other groups [20,29,30], our

results indicate that rBAX promoted opening of the

mPTP, and not only formed supramolecular openings

in the outer mitochondrial membranes, as proposed

previously [31]. rBAX insertion into mitochondrial

membranes induced calcium release from the matrix

space, while cytochrome c content diminished. The

rBAX effect was similar to that exerted by carboxya-

tractyloside when used to induce mPTP opening

(Fig. 1). We also observed that rBAX-induced perme-

ability was totally abolished by the classic mPTP

inhibitor CSA (Fig. 2) and depended on calcium over-

load (Fig. 3).

When the lipid mitochondrial composition was dis-

rupted, a different response was observed. Control

mitochondria showed resistance to the rBAX-inducing

action on the mPTP, following removal of cholesterol

with MbCD. Diminution in cholesterol and GD3 levels

in treated membranes, correlated with low cholesterol

and GD3 content in mitochondrial membranes isolated

from hypothyroid rats that were naturally protected

against mPTP opening induced by rBAX (Fig. 5). It is

conceivable that the cholesterol ⁄ gangliosides ratio may

be relevant to maintaining the structure of the lipid

microdomains, in such a way that disruption of one of

the elements could be translated into a loss of the local

formation of specific and functional raft-associated

complexes. Although the MbCD properties to chelate

cholesterol are well known, there is, to our knowledge,

no report indicating that it also decreases GD3 con-

tent. Indeed, our results show that MbCD induced a

marked decrease in GD3, which represent a novel find-

ing. In this respect, it has been described that MbCD–

cholesterol extraction is accompanied by release of

GM1 molecules and proteins from plasma membranes

[32].

With respect to the relevance of cholesterol in a

mitochondrial lipid raft, it is known that addition of

cholesterol to membrane models containing only phos-

pholipids and sphingolipids permits the formation of a

liquid-order phase in which saturated acyl chains are

highly organized, as in a highly ordered gel phase, but

exhibit lateral mobility more similar to that in the

liquid-ordered crystalline phase [21]. If cholesterol is

the determinant for the generation of a liquid-ordered

phase, it follows that alterations in the cholesterol con-

tent of membranes should lead to changes in their

properties. Our findings suggest that cholesterol

decrease correlated with mPTP opening inhibition in

the presence of rBAX, which is consistent with studies

of cholesterol depletion that alters the function of a

raft-associated potassium channel [33]. Modifications

in the cholesterol content in vivo may lead to altera-

tions in the physical environment of the membrane,

consequently changing the likelihood of certain pro-

teins partitioning into these domains. Furthermore, it

has been reported that hypothyroid conditions affect

the biosynthesis and expression of gangliosides in spe-

cific tissues and cell types [34].

Our findings could be organized into three different

scenarios. First, cholesterol and GD3 in the mitochon-

drial membranes favored rBAX insertion and interac-

tion with the mPTP (control mitochondria); second, a

diminished cholesterol and GD3 content, resulted in a

lower likelihood of raft formation, and hence in cross-

talk disruption between rBAX and PTPm (control

mitochondria + MbCD). This is supported by recent

data indicating that the macromolecular complex

conformed by GD3, the voltage dependent anion chan-

nel-1 (VDAC-1) and the fission protein hFis1 could be

targets in which Bcl-2 family proteins are recruited

[16]. Relevant to this issue is that GD3 and ceramide

have been shown to boost the ability of BAX to

induce the mPTP [20]. Finally, in membranes with

endogenously low cholesterol and ganglioside content

(hypothyroid mitochondria) the closed state of the

mPTP was also favored. It has been reported that

hypothyroidism is associated not only with low choles-

terol and ganglioside levels, but also with decreased

mitochondrial functional activity, oxygen consumption,

ATP synthesis and, relevant for mPTP regulation, with

low cardiolipin content and a diminished amount of

some of the mPTP components (e.g. ANT) [35,36].

The striking difference between rBAX association in

control and hypothyroid mitochondria might be

explained by the differences in cholesterol and ganglio-

side content, along with modifications characteristic of

hypothyroidism, in particular cardiolipin content. A

potential function of GD3 could be to enhance translo-

cation of the pro-apoptotic protein BAX to mitochon-

dria. An attractive hypothesis to explain the results

obtained in hypothyroid mitochondria could be that

cardiolipin is relevant for BAX docking into the mem-

branes. Regarding the relevance of cardiolipin and

mPTP on BAX insertion, there are reports suggesting

that BAX permeabilizes synthetic liposomes only if

cardiolipin is present [31,37] and that oligomeric BAX

does not permeabilize outer membrane vesicles if the



contact sites have been removed [38]. In this sense,

cardiolipin clusters associated with apoptoptic and

energy-flux process proteins have been found at con-

tact sites in the mitochondrial membranes [39].

In conclusion, our results indicate that cholesterol

and GD3 are relevant for the interaction of BAX with

the mPTP. The data provide evidence that specific lip-

ids play a key role in cross-talk between rBAX and the

mPTP. Indeed, more experimental data supporting the

idea that BAX and mPTP converge into mitochondrial

like-raft domains are imperative.

Experimental procedures

Antibodies and reagents

Chemicals were of reagent or higher grade from Sigma-

Aldrich (St. Louis, MO), unless otherwise specified. Glu-

tathione S-transferase (GST)–Sepharose, thrombin prote-

ase and enhanced chemioluminescence system detection

were obtained from Amersham Biosciences (Chalfont St

Giles, UK); protease inhibitors set was from Roche

(Mannheim, Germany) silica gel 60F254 was purchased

from Merck (Darmstadt, Germany); anti-ANT polyclonal

IgG (N-19) was from Santa Cruz Biotechnology Inc.

(Santa Cruz, CA); anti-BAX mAb (Clone 6A7) was from

Alexis Biochemicals (San Diego, CA), anti-(cytochrome c)

mAb (Clone 7H8.2C12) and biotin-conjugated secondary

antibodies were from Zymed Laboratories (San Francisco,

CA).

Induction of hypothyroidism

All animal procedures were performed in accordance with

the Guide for the Care and Use of Laboratory Animals

published by the US National Institutes of Health [publica-

tion no. 85 (23) revised 1996]. Male Wistar rats weighing

380 ± 17 g underwent surgical thyroidectomy with para-

thyroid re-implant, as described previously [40]. Briefly, the

trachea was exposed under anesthesia. Parathyroid glands

were visualized by means of a stereoscopic microscope

(Wild M5, Wild Heerbrugg, Switzerland), dissected from

the thyroid gland and re-implanted into the surrounding

neck muscles. The thyroid gland was then carefully dis-

sected to avoid injury to the laryngeal nerves and com-

pletely excised. The effectiveness of this procedure was

assessed by determining the concentration of calcium, phos-

phorous and thyroxin in 10 sham-operated control and 10

hypothyroid rats, using standard techniques. The results

obtained 15 days after surgery were: Ca2+, 10.2 ± 3 mm in

control versus 10.3 ± 0.2 mm in hypothyroid; phospho-

rous, 6.5 ± 0.3 mm in control versus 6.3 ± 0.5 mm in

hypothyroid and thyroxine 6.4 ± 7 lgÆL)1 in control versus

11.8 ± 1.9 lgÆL)1 in hypothyroid, P < 0.05. The sham

group (375 ± 14 g) underwent a surgical procedure in

which the animals were anesthetized, the trachea was

exposed and the incision was closed, simulating thyroidec-

tomy.

Purification of BAX-DC (rBAX) protein

Recombinant GST–BAX-DC (protein lacking the C-termi-

nal 20 amino acids) was prepared according to Xie et al.

[41] with slight modifications. Briefly, Escherichia coli

BL21(DE3)pLysS cells carrying the plasmid pGEX-4T1–

BAX-DC were grown overnight at 37 �C in Luria Bertani

medium supplemented with 100 lgÆmL)1 ampicillin,

25 lgÆmL)1 chloramphenicol and 1% glucose. Cells were

cultured overnight at 37 �C after induction with 0.4 mm

isopropyl-b-d-thiogalactoside. Harvested cells were dis-

rupted with lysozyme in the presence of 1% TX 100 and

sonicated in NaCl ⁄Pi, pH 7.4. Unbroken cells were elimi-

nated after centrifugation at 5000 g, for 10 min. High speed

centrifugation (100 000 g for 30 min at 4 �C) was per-

formed to clear the supernatant, before glutathione–Sepha-

rose affinity chromatography. Unbound protein was

washed extensively using NaCl ⁄Pi, pH 7.4, supplemented

with 0.1% TX 100 and the protein was recovered by over-

night proteolytic cleavage with thrombin at room tempera-

ture. Eluted rBAX was dialyzed against 250 vol 10 mm

Tris ⁄HCl (pH 8.0), 1 mm EDTA, and 0.1% (v ⁄ v) 2-b-mer-

captoethanol. rBAX was aliquoted and stored at )70 �C.

rBAX oligomerization

rBAX oligomerization was induced by incubating the pro-

tein with 1% octylglucoside for 60 min at 4 �C. The protein

was then diluted 10· in the assay buffer and incubated with

isolated mitochondria, before measurement of mitochon-

drial permeability transition.

Mitochondrial permeability transition pore

opening induced by rBAX

Mitochondria were isolated from rat kidney cortex by dif-

ferential centrifugation in a sucrose-based medium, as

described previously [42]. Mitochondrial protein (1.3 mg)

was incubated for 15 min in the presence of oligomerized

rBAX at the indicated concentration. Opening of the mPTP

was evaluated by measuring mitochondrial calcium move-

ments, mitochondrial swelling and Dw in a basic medium

containing, 125 mm KCl, 10 mm Hepes, 10 mm succinate,

3 mm Pi, pH 7.4, plus 200 lm ADP, 3 lgÆmL)1 rotenone

and, where indicated, 50 lm CaCl2.

Mitochondrial calcium uptake was evaluated spectropho-

tometrically in a double beam spectrophotometer at 675–

685 nm, by using the metallochromic dye Arsenazo III [43].

Mitochondrial swelling was followed by changes in



absorbance at 540 nm and Dw was determined by using the

dye safranine, at 524–554 nm [44].

Measurement of NAD+ content in mitochondria

Mitochondrial NAD+ was measured after perchloric acid

extraction as described previously [45], with minor modifi-

cations. We added 0.5 mL of 21% (v ⁄ v) HClO4 to 10 mg

mitochondrial protein per mL of suspension and incubated

during 30 min in an ice-cold bath. The suspension was cen-

trifuged at 8000 g and the supernatans neutralized. NAD+

was determined fluorometrically at kex ¼ 340 nm and

kem ¼ 460 nm, by measuring NAD+-dependent lactate

dehydrogenase activity, in a medium containing 3 lg of lac-

tate dehydrogenase from rabbit muscle, 400 mm Hydrazine,

500 mm glycine and 10 mm l-lactate pH 9.0, at 25 �C.

rBAX insertion into mitochondria and

cytochrome c release

rBAX insertion and cytochrome c content in mitochondria

were evaluated by western blot analysis, using a primary

anti-BAX mAb (1 lgÆmL)1) or a mAb against cyto-

chrome c (1 : 1000 dilution). Biotin-conjugated secondary

antibodies and streptavidin–peroxidase conjugate were used

followed by an enhanced chemiluminescence system detec-

tion. After maximal calcium release, 1.8 mL of the suspen-

sion was withdrawn and centrifuged at 18 000 g for 10 min.

To discard the protein loosely bound to the membranes,

the mitochondrial pellets were incubated with 0.1 m

Na2CO3, pH 11.5 (alkaline extraction), for 10 min at room

temperature, a procedure that assures that only full inserted

BAX would be detected. Mitochondria were recovered by

centrifugation, washed once with 250 mm sucrose, 10 mm

Tris, pH 7.4, and suspended in sample buffer for electro-

phoresis. Total mitochondrial protein (50 lg) was loaded

into each lane of the SDS ⁄PAGE gels and transferred to

poly(vinylidene difluoride) membranes for immunodetec-

tion. To assess protein loading, the membranes were

‘stripped’ in a buffer containing 62.5 mm Tris ⁄HCl,

100 mm b-mercaptoethanol, 2% SDS, pH 6.7, for 20 min

at 50 �C. The membranes were incubated against anti-ANT

or anti-VDAC polyclonal IgG.

Cholesterol and ganglioside measurements

Control and hypothyroid mitochondria were incubated with

the cholesterol-chelator MbCD for 25 min at 25 �C and

mild shaking. After depletion, mitochondria were centri-

fuged at 8000 g and suspended in NaCl ⁄Pi, pH 7.4, for

further analysis. Total cholesterol was determined from

mitochondrial lipid extracts obtained with chloro-

form ⁄methanol (2 : 1) in the presence of 0.02% butyl-

hydroxy-toluene and using 50 lg stigmasterol as the

internal standard, as described previously [46]. Lipid

extracts were incubated overnight at room temperature with

hexamethyldisilazane and trimethylchlorsilane in dry pyri-

dine. Cholesterol was purified in a fused silica noncapillary

column SE 54 (30 cm, 0.35 mm id) and quantified by gas

chromatography using a gas chromatograph (Carlo Erba

2003), equipped with a flame ionization detector and a

split-less inlet system.

Gangliosides were extracted from control and hypo-

thyroid mitochondria previously treated with MbCD and

analyzed by preparative TLC on silica gel plates with chlo-

roform ⁄methanol ⁄ 0.2% CaCl2 ⁄ acetic acid (7 : 3 : 0.5 : 0.2

v ⁄ v ⁄ v ⁄ v), as described previously [47]. The plates were

sprayed with 10% H2SO4 (v ⁄ v) and heated for spot detec-

tion. GD3 standard gave two equivalent spots (RF ¼ 0.85

and 0.81). The double band is known to result from the

heterogeneity of fatty acid composition in gangliosides.

Positive signal in the samples correlated with the band pre-

senting a retention factor of 0.85 from the GD3 standard.

No signal was detected comigrating with the GM1 stan-

dard. Alternatively, gangliosides were specifically detected

with resorcinol reagent [48] and compared with the signal

developed by H2SO4 using a simple ‘dot’ assay. Cardiolipin

content was analyzed after inner mitochondrial phospho-

lipid extraction with chloroform ⁄methanol (2 : 1 v ⁄ v), thenindividual phospholipids were separated by TLC on silica

gel plates with chloroform ⁄methanol ⁄H2O (65 : 25 : 4

v ⁄ v ⁄ v) and visualized with iodine. Spots comigrating with

the cardiolipin standard were scraped out from the chro-

matogram and quantified spectrophotometrically at

472 ⁄ 492 nm using acridine orange, as described [49].

Statistical analysis

Values are given as means ± SD and were evaluated by

one-way ANOVA. P < 0.05 was considered the threshold

for statistical significance between the groups indicated.

Acknowledgments

We greatly acknowledge the generous gift of pGEX-

4T-I BAX plasmid from Dr John Reed (Burnham

Institute for Medical Research, La Jolla, CA) and par-

tial support from the Doctorate Program in Biomedi-

cal Sciences of the National Autonomous University

of Mexico (UNAM) to Eduardo Martınez-Abundis.

We also thank Mr Jose Santamarıa (Nephrology

Department, National Institute of Cardiology, Ignacio

Chavez) for performing the surgical procedures. This

study was partially supported by grant 46456-M to CZ

from the National Council of Science and Technology

(CONACYT), Mexico. The authors state no conflict

of interest.



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Changes in specific lipids regulate BAX-induced mitochondrial permeability transition