gtp-binding proteins in adrenocortical mitochondria

ELSEVIER Biochimica et Biophysica Acta 1248 (1995) 159-169

Btt Biochi~ic~a et Biophysica Acta

GTP-binding proteins in adrenocortical mitochondria

Murray Thomson 1, Maxine Korn, Peter F. Hall *

Department of Endocrinology, Prince of Wales Hospital, Randwick, NSW 2031, Australia

Received 29 March 1994; accepted 25 November 1994

Abstract

We have identified two GTP-binding proteins in mitochondria from bovine adrenal cortex (fasciculata). Sub-mitochondrial particles were fractionated into inner membrane, contact point and outer membrane vesicles on sucrose density gradients. These sub-mitochondrial fractions were identified by the presence of enzyme markers and electron microscopy. Photoaffinity labelling with [T-32p]GTP identified a 45 kDa GTP-binding protein in outer mitochondrial membranes and a 19 kDa protein in the contact points. The molecular weight of 45 kDa and requirement for Mg 2+ ions raise the possibility that this protein is an a subunit of a heterotrimeric GTP-binding protein or a novel GTP-binding protein. The specificity of nucleotide binding, the requirement for low concentrations of Mg 2+ (0.1 raM) and molecular weight of 19 kDa suggest that this protein is a typical member of the so-called small GTP-binding protein family. The location of 45 kDa in the outer membrane and that of 19 kDa in the contact points suggest roles for these proteins in the interaction with the extramitochondrial environment and in the regulation of mitochondrial membranes, respectively.

Keywords: GTP-binding protein; Adrenal cortex; Mitochondrion; Photoaffinity labeling; Cholesterol

I. Introduction

In the adrenal gland mad other steroidogenic tissues, the synthesis of steroid honnones begins in the inner mitochondrial membrane with the conversion of cholesterol to pregnenolone (side-chain cleavage of cholesterol) cat- alyzed by the enzyme cytochrome P-450 side-chain cleavage (P-450sc ¢) [1-3]. The rate-limiting step for this reac- tion appears to involve the transport of cholesterol from storage droplets in the cytoplasm to the mitochondria and from the outer membrane to P-450sc c in the inner mitochondrial membrane. This transport is stimulated by adrenocorticotropic hormone (ACTH) via cAMP [4,5]. The mechanism of cholesterol transport from the outer to the inner membrane is known to require synthesis of new protein [6,7]. Recently a small protein called endozepine (also known as benzodiazepine-binding inhibitor) has been isolated from bovine fasciculata and has been shown to

Abbreviations: ACTH, adrenocorticotropic hormone; P-450see, cytochrome P-450 side chain cleavage.

* Corresponding author. 1 Present address: Department of Biological Sciences, University of

Western Sydney, Nepean, Westmead North, NSW 2145, Australia.

0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 4 ) 0 0 2 3 4 - 7

accelerate the movement of cholesterol from outer to inner membrane [8,9]. On the other hand it is known that in contrast to cholesterol, hydroxylated derivatives of cholesterol pass freely from outside the mitochondrion to e-450sc c and are rapidly converted to pregnenolone in the absence of ACTH [10]. In addition, the slow transport of cholesterol to the interior of the mitochondrion and P-450~c is in marked contrast to the rapid passage of this substrate to the enzyme in mitochondria from which the outer membrane has been removed (so-called mitoplasts) [11]. Evi- dently the outer mitochondrial membrane provides a barrier to the passage of.cholesterol.

It has been reported that GTP increases the synthesis of pregnenolone in adrenocortical mitochondria [12]. These authors have suggested that the nucleotide may promote the translocation of cholesterol to inner mitochondrial membrane. Moreover, it is now clear that GTP is often involved in intracellular transport and in the fusion of membranes in various cells (reviewed in [13]). GTP com- monly acts via GTP-binding proteins and a recent report describes the occurrence of a 52 kDa GTP-binding protein in hepatic mitochondria [14]. It was therefore decided to determine whether GTP-binding proteins occur in adrenal mitochondria, and if so, to determine the sub-mitochondrial location of these GTP-binding proteins.

160 M. Thomson et al. / Biochimica et Biophysica Acta 1248 (1995) 159-169

2. Materials and methods

2.1. Materials

['y-32p]GTP (specific activity 6000 Ci/mmol; 10 mCi//xl) was supplied by Du Pont (Sydney, Australia) and was diluted 1:10 with Tris-HCl buffer (40 mM, pH 7.4) to a final concentration of 0.17/xM. The silver stain kit (Silver Stain Plus) was from Bio-Rad (Sydney, Aus- tralia).

2.2. Methods

Isolation of mitochondria and subcellular fractions from bovine adrenal fasciculata. Bovine adrenal glands (five) were collected from the abattoir in ice-cold phosphate- buffered saline (PBS). Fasciculata was dissected from glomerulosa with a scalpel and homogenized in 250 ml buffer A (Tris-HCl 20 mM, mannitol 210 mM, sucrose 70 mM) using a motor-driven teflon pestle (Type 853 304/0, Braun, Australia). Mitochondria were prepared by the method of Adams et al. [15]. Briefly, the homogenate was centrifuged at 700 × g at 4 ° C for 10 min and the supernatant was kept at 4 ° C. The pellet was re-extracted twice with 200 ml buffer A. The pellet containing the plasma membranes was kept at - 7 0 ° C for further studies. The supernatants were pooled and centrifuged at 9750 × g for 15 min at 4 ° C and the mitochondrial pellet collected. The pellet was then resuspended with a glass pestle in 200 ml buffer A and centrifuged at 9750 × g at 4 ° C. This step was performed three times to give the final mitochondrial pellet. In some studies mitochondria were further purified by sedimentation in a Percoll gradient [16]. Microsomes and plasma membranes were isolated by methods described previously [17,18].

Isolation of submitochondrial fractions. Inner and outer membranes and contact points were prepared from whole mitochondria by a modification of the method of Adams et al. [15]. Mitochondria were subjected to swelling and shrinking by incubation in 15 ml of 10 mM phosphate buffer (pH 7.4) at 4 ° C for 20 min, followed by the addition of 6 ml 60% sucrose and incubated for 20 min. Mitochondrial membranes were then further disrupted by a Soniprobe Type 7530 A Sonifier (Dawe Instruments, Eng- land) three-times for 30 s at level 6. Unbroken mitochondria were removed by centrifugation at 9750 × g for 20 min at 4 ° C and 5 ml aliquots of supernatant were layered on a linear sucrose gradient (24.4-61.2% in a total volume of 23 ml). Gradients were subjected to ultracen- trifugation at 4 ° C for 20 h at 100000 × g. The gradient was then collected as 20 fractions of 1.4 ml each starting at the bottom of tube. This method separates inner membrane which is found at the bottom of the tube, outer membrane which is located towards the top of the tube and contact points which are located between inner and outer membrane [15].

Enzyme assays. The activity of cytochrome oxidase (EC 1.9.3.1; inner membrane) was assayed by the method of Cooperstein and Lazarow [19], Rotenone insensitive NADH-dependent cytochrome-c reductase (EC 1.6.2.2; outer membrane) was measured according to Sottocasa et al. [20]. Hexokinase (EC 2.7.1.1; contact points) and creatine kinase (EC 2.7.3.2) were measured according to Bucher

1

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1

o <

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C

1-

I-- O <

,,, 0.5.

--J W if-

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i i I I I I I I I I I I I I I I I I

FRACTION NUMBER

Fig. 1. Separation of sub-mituchondrial fractions from bovine fasciculata. Sonicated mitochondria were centrifuged into a sucrose density gradient and 20 fractions (1.4 ml) were collected from the bottom of the tube. Aliquots of each fraction were used to measure the following enzymes: (A) cytochrome oxidase (inner membrane), (B) hexokinase (contact points), (C) rotenone-insensitive NADH-dependent cytochrome-c reductase (outer membrane), (D) creatine kinase. For each graph values are shown as a fraction of the highest value. Three experiments were performed (each using membranes from a separate preparation of mitochondria) with similar results.

M. Thomson et aL /Biochimica et Biophysica Acta 1248 (1995) 159-169 161

et al. [21]. Al l enzyme assays were performed under

condi t ions of Vma X.

Photoaffinity labelling o f adrenal mitochondria and submitochondrial fractions. Photoaff ini ty label l ing was

performed by a modif icat ion of the technique reported by

Li thgow et al. [14] as follows. Samples ( 2 / x l ) were mixed with buffer salts and ['g-32 P]GTP, so that the cross- l inking

buffer consisted of 20 m M Hepes, 0.5 m M EDTA, 5 m M

contact points

M W 2 4 6 8 10 12 14 16 18 2 0

inner A membrane

92"5 -.

6 9 "'

4 6 -' 3 0 -.

21'5- .

14~3-,

outer membrane

B MW

9 2 . 5 -

6 9

4 6

3 0

21.5 -

14.3 -

2 4 6 8 10 12 14 16 18 20

C

D

.>~

MW

9 2 " 5 -

6 9 - 4 6 -

3 0 -

21"5-

14"3-

1-

0.5 ̧

2 4 6

4 I I I I I ' l " I J I I I ( I I I I I I

10 12 14 16 18 2 0

E 1-

>

0.5

I I I I [ I I I L I I I I I I

Fig. 2. Polyacrylamide gel electrophoresis and GTP binding of sub-mitochondrial fractions. (A) Gel electrophoresis of fractions from a sucrose gradient stained with silver. Fractions from the gradient are indicated by numbers corresponding with those in Fig. 1. (B) Distribution of GYP-binding proteins demonstrated by UV cross-linking of proteins in aliquots from the sucrose gradient with [1,-32p]GTP (5 mM MgC12) followed by autoradiography. (C) As for (B) except that MgCI 2 concentration was 0.1 raM. Densitometry values for the 45 kDa bands in (B) and the 19 kDa bands in (C) are shown in (D) and (E), respectively. Three experiments were performed (each using membranes from a separate preparation of mitochondria) with similar results.

162 M. Thomson et a l . / Biochimica et Biophysica Acta 1248 (1995) 159-169

MgCI 2 (unless otherwise stated) and [7-32p]GTP (0.17 pmol, 1 mCi per sample). The total volume was 25 /xl. Binding was carried out in 96 well microtitre plates on ice for 10 rain. Samples were then irradiated with UV light for 2 min using a UV transilluminator (TF 35M, France, 6 × 15 W tubes, 312 nm) inverted over the samples at a distance of 1 cm. Samples were then prepared for gel electrophoresis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). One- and two-dimensional (iso- electric focusing as the first dimension) gels were performed using the Bio-Rad Mini Protean II system. Gels were stained with silver using a commercially available kit. Gels were dried and subjected to autoradiography for 2-48 h at - 80 ° C, so that the intensity of each image with respect to time was in the linear range. When necessary, the intensity of the images on the autoradiogram were measured by scanning densitometry using a Hoefer (USA) scanning densitometer.

Transmission electron microscopy. Inner (pooled fractions 3 -6 from 3 sucrose density gradients), outer membranes (pooled fractions 14-18) and contact points (pooled fractions 8-13) were dialyzed against 10 mM phosphate buffer at 4 ° C for 1 h with 4 changes of buffer. The

dialysed fractions were then centrifuged at 200 000 × g at 4 ° C for 30 min and the pellets prepared for electron microscopy. The three samples were fixed as described in detail previously [22]. Thin sections were stained with uranyl acetate and lead citrate and examined under a Hitachi 7000 electron microscope. Contact point vesicles were counted in each of 3 fields of 25/~m 2 in each of the three fractions.

Treatment of contact points with trypsin. Contact points prepared by sucrose density gradient were pooled (fractions 8-13) and diluted with two volumes of buffer B (20 mM Hepes, 100 mM NaC1 and 2 mM MgC12) at 4 ° C. Samples were centrifuged at 200000 × g for 30 min at 4 ° C and the pellet was resuspended in buffer B at a concentration of 0.5 mg protein/ml. Aliquots (100 /.d) of the contact point suspension were incubated with various concentrations of trypsin for 15 min at 4 ° C. Reactions were terminated by the addition of excess soy bean trypsin inhibitor.

Protein assay. Protein was measured using the Pierce BCA protein assay kit according to the instructions of the manufacturer.

Statistical analysis. Statistical analysis was performed by one way analysis of variance (ANOVA). A P value of < 0.05 was accepted as significant.

MW

lane 1 2 3 4 5

92 "5 -

6 9 -

4 6

3 0 -

2 1 , 5 -

1 4 ' 3 -

A

MW

9 2 . 5 - -

6 9 --

4 6 - -

3 0 - -

2 1 . 5 - -

14 .5 - - "

lane 1 2 3 4 5

i . . . . i ~ i~!! !!ii . . . .

B Fig. 3. Comparison of the effects of Mg 2+ and Mn 2+ on GTP binding to 45 kDa and 19 kDa proteins. UV cross-linking was performed with mitochondrial membranes (2 m g / m l protein) and the following concentrations of Mg 2+ (A) or Mn 2+ (B); 0 (lanes 1), 0.1 mM (lanes 2), 2.5 mM (lanes 3), 5.0 mM (lanes 4) and 10.0 rnM (lanes 5). Samples were subjected to gel electrophoresis followed by autoradiography. Three experiments were performed (each using membranes from a separate preparation of mitochondria) with similar results.

3. Results

3.1. Preparation of submitochondrial fractions by sucrose density gradient sedimentation

Submitochondrial fractions from bovine adrenal were prepared on sucrose density gradients in a manner similar to that used by other workers for mitochondria from rat liver, brain and kidney [15]. Marker enzymes were used to calibrate the gradient. Cytochrome oxidase (inner membrane) activity was found in high density fractions (fractions 3-9) with some skewing towards the bottom of the gradient (Fig. 1A). Rotenone-insensitive NADH-dependent cytochrome-c reductase (outer membrane) was found close to the top of the gradient (fractions 14-18; Fig. 1C). Hexokinase (contact points) was found at approximately the middle of the gradient (fractions 6-13; Fig. 1B). The distribution of creatine kinase was found to be very similar to that for rotenone-insensitive NADH-dependent cytochrome-c reductase activity and therefore appears to be mainly associated with the outer mitochondrial membrane, although some activity is present in the contact point fractions (Fig. 1D).

3.2. Photoaffinity labelling of submitochondrial fractions

Fractions from the sucrose density gradients were subjected to photoaffinity labelling with [7-32p]GTP (using cross-linking buffer containing 5 mM MgC12) followed by

M. Thomson et al. / Biochimica et Biophysica Acta 1248 (1995) 159-169 163

SDS-PAGE. A typical ~:cl stained with silver is shown in Fig. 2A. Autoradiography revealed a GTP-binding protein of = 45 kDa in low density fractions corresponding to outer membrane (Fig 2B). A second fainter band with an apparent MW of 19 kDa~, was observed in the intermediate (contact point) region of the autoradiogram. When UV cross linking was perfoimed in 0.1 mM MgC12 the intensity of labelling of the 19 kDa bands was increased as shown in Fig. 2C. Densiitometry of the 45 kDa bands seen in Fig. 2B are shown in Fig. 2D. Intensity of labelling of the 19 kDa bands (Fig. 2C) closely coincides with values for hexokinase activity (Fig. 1C). Densitometry of the 19 kDa bands is shown in Fig. 1E. The distribution of 19 kDa strongly suggests that it is associated with contact points. Similar results (not shown) were observed when mitochondria were further purified on a Percoll gradient [16] before sonication.

3.3. The influence o f magnesium and manganese ions on photoaffinity labelling of 45 kDa and 19 kDa

Samples of membrane were incubated in photoaffinity labelling buffer containing final concentrations of 0, 0.1, 2.5, 5.0 and 10.0 mM MgCl 2. This experiment demon-

strated that Mg 2+ is required for binding of [7 -32 P]GTP to both 45 and 19 kDa and that the optimum concentration of Mg 2+ for the two proteins is very different. The 45 kDa protein bound appreciable amounts of [~-32p]GTP at a concentration of 2.5 mM Mg 2+ (Fig. 3A). Increasing the concentration of Mg 2÷ did not diminish binding. Similarly for MnCl 2 (no MgC12 in cross linking mixture) concentrations of 2.5 mM or more greatly increased binding of [7-32p]GTP to 45 kDa (Fig. 3B). By contrast, 19 kDa showed binding for [?/_32 P]GTP at a concentration of 0.1 mM Mg 2+ (Fig. 3A). Very little or no binding was seen with no Mg 2+. Concentrations of MgC12 higher than 0.1 mM decreased binding of [y-32p]GTP to 19 kDa (Fig. 3A). It was apparent therefore that either 45 kDa or 19 kDa can be preferentially identified by photoaffinity labelling in buffer containing MgCI 2 at a concentration of 2.5 mM or 0.1 mM respectively. While both GTP-binding proteins are identified by photoaff'mity labelling in either 2.5 mM or 0.1 mM MgC12 in some studies the concentration of MgC12 was chosen to intensify the labelling of one or other of the two GTP-binding proteins.

Manganese chloride did not support [7- 32 P]GTP binding to 19 kDa, however, binding to 45 kDa was increased by 2.5, 5.0 and 10.0 mM MnC12 (Fig. 3B).

AMP ADP ATP GMP GDP GTP

O O O O

<

P

03

Q.

C 70"

60'

50"

40'

30'

20'

10'

~ ADP • GDP

=k

0 1(} 100

D 70"

60"

50"

40"

30"

20'

10'

0

. ~ o . - - AT P • GTP

0 100 excess of unlabled nucleotide excess of unlabled nucleotide

Fig. 4. Specificity of ['y-32p]GTP binding to 45 kDa. The 45 kDa protein was cross-linked to ['y-32p]GTP as described in Section 2, in the presence or absence of a ten- or one hundred-fold excess of various non radioactive nucleotides as indicated. Samples were analysed by gel electrophoresis followed by autoradiography as shown in (A) and (B). Tluee experiments were performed on three different preparations of mitochondria with similar results. Peak areas for ADP and GDP are shown in (C), peak areas for ATP and GTP are shown in (D). Peak areas represent values for three experiments each performed on membranes from a separate preparation of mitochondria (mean + S.E.) determined by densitometry. * Significant difference from control, P < 0.05.

164 M. Thomson et al./ Biochimica et Biophysica Acta 1248 (1995) 159-169

3.4. Specificity of GTP binding to 45 kDa and 19 kDa

Fig. 4 shows photoaffinity labeling of 45 kDa with [T-32p]GTP with and without competing unlabelled nucleotides. Autoradiograms from photoaffinity labelling of this GTP-binding protein are shown in Fig. 4A and 4B. Densitometry of these bands with the diphosphates (ADP and GDP) and triphosphates (ATP and GTP) are shown in 4C and 4D respectively. Monophosphates (AMP and GMP) were without effect on binding to [~/-32p]GTP. Both the diphosphates (GDP and ADP) significantly inhibited binding of [7-32p]GTP at 10 × and 100 × the concentration of the labelled nucleotide. Both the triphosphates (GTP and ATP) significantly inhibited binding at 100 × the concentration of the labelled nucleotide.

Fig. 5 shows that with 19 kDa the monophosphates were without effect on photoaffinity labelling. At 10-fold excess only GDP and GTP displaced the label showing that 19 kDa is specific for guanine nucleotides. ATP and ADP inhibited binding at 100-fold excess but to a much lesser extent than GDP and GTP. The studies on 19 kDa were performed with 0.1 mM MgC12.

3.5. Effect of KCl on [T-32p]GTP binding by 45 and 19 kDa proteins

The effect of K + ions on the binding of ['y-32p]GTP to 45 kDa and 19 kDa was investigated by performing the

A 1 2 3 4 5 6

B 1 2 3 4 5 6

Fig. 6. Effect of K + on binding of [7-32p]GTP to 45 and 19 kDa proteins. The experiments were performed as described under Fig. 3 with 5 mM MgC12 (A) and 0.1 mM MgC12 (B) with and without the following concentrations of KC1, 0 (lanes 1), 50 mM (lanes 2), 150 mM (lanes 3), 300 mM (lanes 4), 500 mM 0anes 5) and 1000 mM (lanes 6). Samples were analysed by gel electrophoresis followed by autoradiography. Three experiments were performed (each using membranes from a separate preparation of mitochondria) with similar results.

UV cross-linking procedure on membranes in the absence and presence of various concentrations of KC1. The labelling of both proteins was clearly inhibited by 500 and 1000 mM KC1 (Fig. 6). Dialysis of membranes following incubation reversed this inhibition (not shown). Replicate determinations showed that binding of [y_32p] in 50 and 150 mM was not significantly different from values without KCI.

AMP ADP ATP

A ~ o ~ ~ B GMP GDP GTP

0 0

Q,.

C 70"

60"

50"

40"

30"

20"

10'

0

• GDP X ~ . ,

o 1'o

D 70

60

50

40

30

20

10

~ ATP

10 100

excess of unlabeled nuc|eotide excess of unlabled nucleotide

Fig. 5. Specificity of [y-32p]GTP binding to 19 kDa. This experiment was performed as described under Fig. 4, except that proteins were cross-linked in the presence of 0.1 mM MgCI2, *P < 0.01.

M. Thomson et al. / Biochimica et Biophysica Acta 1248 (1995) 159-169 165

MW

9 2 " 5 -

6 9 -

4 6 -

3 0 -

21 "5 -

1 4 - 3 -

4'5

pl 5'0 5 .5 6-0 6"3 8 '4

pl 4"5 5 . 0 5 .5 i m l i #

m w , , , 6-0 6.3 8.4

I I

9 2 " 5 -

6 9 -

4 6 - D

3 0 -

21 " 5 -

1 4 . 3 -

B

A

Fig. 7. Two-dimensional electrophoresis of mitochondrial proteins. Fol- lowing UV cross-lialdng of mitochondrial membranes (2 /zl; 2 mg /ml protein each) samples were subjected to two-dimensional gel electrophoresis. Gels were stained with silver (A) and subjected to autoradiography (B). Three experiments were performed (each using membranes from a separate prepar~.tion of mitochondria) with similar results.

3.6. Two-dimensional electrophoresis of 45 kDa and 19 kDa

Two-dimensional electrophoresis (gel stained with silver shown in Fig. 7A) followed by autoradiography was used to determine the homogeneity of the two bands at 45 kDa and 19 kDa and to estimate pL A single spot corresponding to a MW of 45 kDa with an apparent p I of 7.1 was seen on autoradiography (Fig. 7B). A second spot was observed corresponding to a MW of 19 kDa with an apparent pI of 8.9 (Fig. 7B). The spots corresponding to 45 kDa and 19 kDa were very slightly oblong in shape because the 2D gel is oblong and the proteins spots are therefore longer in the first dimension, than they are in the second dimension. The spots are of smooth circumference which is indicative of a single protein and we have never seen evidence of 45 kDa and 19 kDa spots made up of multiple proteins with similar pI values. Multiple protein spots on 2D gels do not have a smooth circumference they exhibit a caterpillar shape. There was no detectable stain- ing seen on the gel corresponding to the positions of 45 kDa and 19 kDa on the autoradiograph.

3. 7. Electron microscopy of submitochondrial fractions

Thin sections of three samples were examined. Inner membrane fractions showed mainly sheets of membrane and single ring structures (results not shown). As previously reported, outer membrane fractions showed vesicles (single ring structures) [23] (results not shown). The contact point fraction contained a large number of double ring

A B

4 0 0 -

¢q

E 300 tr) ¢,4

200 O Q .

c o 1 0 0 o

i nne r con tac t p o i n t s o u t e r

fraction

Fig. 8. Electron microscopy of contact points. (A) Thin sections were prepared from a pellet of contact points as described under Section 2. Bar: 50 nm, arrow: contact point. (B) Histogram showing the number of double ring structures (contact points) in the inner membrane, outer membrane and contact point fractions, + S.E., n = 3.


structures (Fig. 8A) In some of the double ring structures the plane of section passed through the junction between outer and inner membrane making the contact point clearly visible (Fig 8A arrow). The distribution of these structures in the three fractions shows that they were virtually confined to the contact point fractions (Fig. 8B).

3.8. The action o f trypsin on contact points

~.~-, rb v ,O ~

1 2 3

Contact point vesicles consist of fragments of inner and outer membranes joined at the contact point proper (see this section and refs. [15] and [27]). The location of 45 kDa in the outer membrane and 19 kDa in the contact points was confirmed by the action of trypsin on contact point vesicles. When contact point vesicles were incubated with increasing concentrations of trypsin the intensity of binding of the labelled nucleotide to 19 kDa was not affected. On the other hand binding of [T-32p]GTP to 45 kDa was completely lost with trypsin (Fig. 9A). Photoaffinity la-

A MW

69 _ 46 -

30 -

21 "5 -

14.3-

lane

1 2 3 4

U

B 1.1

1,0

• I I 0 , 9 -

,> 0.8 -

¢~ 0 .7 - - \ ~ cytochrome oxidase

_~ 0.6 kinase ,=

0.5

0.4

0.3 , , , 0 2 0 4 0 6 0

t rypsin (~g/mg protein)

Fig. 9. Action of trypsin on binding of [T-32 P]GTP to 45 kDa and 19 kDa proteins. (A) Contact points were treated with various concentrations of trypsin. Aliquots of the same samples as those used for electrophoresis (A), were used to measure enzyme activities (Section 2). Three experiments were performed (each using membranes from a separate preparation of mitochondria) results show the mean + S.E.

4 5 k D . - - ~

1 9 k D - - ~ ~ ~

Fig. 10. Photoaffinity labelling of adrenal cell mitochondria and sub-cellular fractions. (A) Plasma membranes, (B) mitochondria and (C) microsomes. Photoaffinity labelling was performed as described under experi- mental procedure. Samples were subjected to gel electrophoresis followed by autoradiography. Three experiments were performed (each using membranes from a separate preparation of mitochondria) with similar results.

belling was performed with 0.1 mM MgCI 2 (Fig. 9A) and 2.5 mM MgCI 2 (results not shown) with similar results. However, both 45 kDa and 19 kDa proteins are susceptible to tryptic digestion when solubilized (membranes were extracted in 5% Triton X-100, 0.5 M KC1, 10 mM Tris-HC1 (pH 7.4), then the buffer was exchanged for 0.5% Triton X-100, 10 mM Tris-HC1 (pH 7.4) and exposed to trypsin (60 /xg /mg protein and digests examined on SDS polyacrylamide gels, data not shown). Moreover, trypsin clearly attacked the outer membrane marker rotenone-insensitive NADH-dependent cytochrome-c reductase, but had little effect on the inner membrane marker cytochrome oxidase or the contact point marker hexokinase (Fig. 9B). Evi- dently trypsin is excluded from the interior of the contact point vesicles so that loss of rotenone-insensitive cytochrome-c reductase activity and 45 kDa binding indi- cates that these two proteins are exposed to the extra-mitochondrial medium (containing trypsin) in contrast to 19 kDa.

3.9. Photoaffinity labelling o f adrenal cell membranes and microsomes

To determine the distribution of GTP-binding proteins within the fasciculata cell, plasma membranes, mitochondria and microsomes prepared from fasciculata cells were suspended in an equal volume of buffer A and 2 /xl samples were cross-linked to [y-32 P]GTP (as in Section 2) followed by gel electrophoresis and autoradiography (Fig. 10). Microsomes, mitochondria and plasma membrane all

M. Thomson et al. /Biochimica et Biophysica Acta 1248 (1995) 159-169 167

contain GTP-binding proteins of MW 19 kDa. Plasma membrane and microsomes displayed traces of GTP-binding corresponding to MW 45 kDa.

The relative intensities of the signals corresponding to 45 kDa and 19 kDa are quite different for GTP-binding proteins from the plasma membrane and microsome fractions compared to those from mitochondria. For example the 45 kDa signal is intense in mitochondria but faint in plasma membrane and microsome fractions.

4. Discussion

The present studies have identified two mitochondrial GTP-binding proteins in bovine adrenocortical fasciculata cells. The mitochondria used to detect these proteins were purified by an established method [15]. In some experiments the mitochondria were further purified using a Per- coil gradient which has been shown to remove trace amounts of contaminating proteins [16]. The mitochondrial membranes were purified in a sucrose gradient. The mix of the two GTP-binding proteins in the microsomes and plasma membranes is quite different from that seen in mitochondria thereby excluding these structures as a contaminating source of the two GTP-binding proteins reported here. In particula:r 45 kDa is poorly represented in microsomes and plasma membranes relative to 19 kDa. The 45 kDa mitochondrial protein may have a higher affinity for [32 P]GTP and/or cross link to the GTP ligand more effectively than the G proteins in the plasma membrane. The location of 1 c~ kDa in contact points and failure of trypsin to attack the protein in these structures also makes it unlikely that thiis protein is associated with mitochondria as the result of contamination by other cellular membranes. Although a 19 kDa protein is present in both plasma membranes and microsomes, only future studies will determine whether these 19 kDa proteins are the same as that found in mitochondrial contact points. The same applies to the 45 kDa protein observed in trace amounts in plasma membranes. It is entirely possible, however, that both GTP-binding proteins are located in various parts of the cell, especially since., other GTP-binding proteins are known to undergo intracellular translocation [24]. The 45 kDa and 19 kDa mitochondrial GTP-binding proteins may be tissue specific to adrenocortical cells as they are not observed in mitochondri,t from liver [14].

The technique of affinity labelling with [32p]GTP is now used [25,14] and has been well characterized [26]. It has been shown that without UV light, labelled GTP attaches to some GTP-binding proteins by tight, but non- covalent binding that is not disrupted by SDS PAGE and some GTP-binding proteins do not need to be cross linked to GTP by UV light [26]. In other cases UV cross linking is needed to label some GTP-binding proteins [26]. In the present study it was fourLd that 45 kDa and 19 kDa could be labelled without UV c:ross linking, although exposure to

UV light markedly intensified the signal (results not shown). Different GTP-binding proteins may vary in the requirement for UV cross linkage to GTP and this may reflect structural differences in the GTP-binding protein family [26].

The occurrence of 45 kDa in the outer membrane and 19 kDa in contact points was established by means of gradient sedimentation of submitochondrial fractions. The positions of outer and inner membrane and contact point fractions in the gradient were demonstrated using marker enzymes and the distribution of these enzymes (with the exception of creatine kinase) gave profiles consistent with those from other types of cells [15]. Slight differences in the methods of preparing mitochondria, as well as the difference in species and tissue origin may explain the occurrence of creatine kinase mainly in the outer membranes of bovine adrenocortical mitochondria, whereas Adams et al. [15] find this enzyme mainly in the contact point fraction of mitochondria from rat brain and liver. Contact point fractions are believed to be composed of two concentric vesicles joined at a contact point. [15]. Such structures are clearly seen in fractions from bovine adrenal cortex by electron microscopy (Fig. 8). These two ring structures are almost entirely confined to the contact point fraction defined by the presence of hexokinase after gradient sedimentation. Studies by Adams and colleagues (reviewed in [27]) suggest that the outer membrane of the vesicle is composed of outer membrane. This conclusion is supported by the present results since the inner membrane enzyme marker, cytochrome oxidase is not attacked by trypsin. Clearly the vesicles are sealed and of the three marker enzymes, only rotenone-insensitive NADH-dependent cytochrome reductase is accessible to external trypsin. Moreover, contact point vesicles contain hexokinase activity but because of the structure of contact point vesicles it is to be expected that these fractions also possess some outer and inner marker enzyme activities as shown in the present studies.

The 45 kDa protein is of a molecular weight consistent with that of an tz-subunit of the heterotrimeric GTP-binding protein family (between 39 and 52 kDa) which in- cludes many proteins that act as signal transducers from membrane receptors to effectors such as adenylate cyclase, phosphoinositide-specific phospholipase C and potassium channels [24]. It is not yet clear, however, whether 45 kDa belongs to this GTP-binding protein sub family. It is well known that Ga subunits bind GTP (active form) and GDP (inactive form) [24]. Moreover, 45 kDa binds GTP and GDP but not GMP as is expected for a Ga protein. However, the specificity for guanosine di- and triphosphates is not absolute, since ADP and ATP also bind to 45 kDa. The equipotency of ATP and GTP in competing with ['y-32p]GTP for binding to 45 kDa is not considered a typical characteristic of a Gc~ protein. However, it is becoming clear that many GTP-binding proteins do not have absolute specificity for GTP and GDP. ATP has been


shown to inhibit [32p]GTP binding to a Gc~ protein, albeit by less than 30% [25]. In addition some nuclear GTP-binding proteins [25] and several other GTP-binding proteins [28-30] have been shown to bind adenine nucleotides in addition to GTP. The 45 kDa protein may bind both GTP and ATP in vivo as has been proposed for GTP-binding proteins in rat nuclear envelopes [25].

The location of 45 kDa is significant, since it is found mainly in the outer mitochondrial membrane. Moreover, treatment with trypsin established that the GTP binding site is exposed to the extramitochondrial environment. The location of 45 kDa in the outer membrane is in keeping with a role for the protein in binding GTP and possibly proteins such as GTP-exchange proteins, from the cytoplasm. It is interesting that 45 kDa was not detected in the inner mitochondrial membrane suggesting that this protein does not act as a carrier for intramitochondrial transport of one or more substances to the inner membrane.

The 19 kDa protein appears to belong to a second group of GTP-binding proteins which are monomeric and are referred to as small GTP-binding proteins due to their relatively low molecular masses (18-36 kDa) [13]. The small GTP-binding proteins include the Ras p21 family. Like heterotrimeric GTP-binding proteins, small GTP-binding proteins are inactive when bound to GDP but are activated in the GTP bound state and 19 kDa clearly displays specificity in binding for GDP and GTP, among the nucleotides tested. As is the case with other small GTP-binding proteins, Mg 2+ stimulates GTP binding at low concentrations [31] with saturation occurring at around 0.1 mM; higher concentrations inhibit binding, while Mn 2+ is without effect. As with other GTP-binding proteins, high concentrations of K + inhibit GTP-binding [32]. This high ionic strength inhibition is non-specific and reversible since dialysis of membranes incubated with 500 mM K + leads to the return of GTP-binding activity with both 19 and 45 kDa proteins (results not shown). Other workers have used 500 mM KCI to aid detergent extraction of membranous G-proteins [32] and preliminary studies show that this approach is useful for 45 kDa and 19 kDa.

The location of 19 kDa protein in contact points is likely to be important. Consistent with this location of the protein is the finding that the GTP binding region is shielded from the surrounding medium, since treatment with trypsin was without effect on the GTP-binding activity of this protein. Contact points between outer and inner mitochondrial membranes are thought to be transient structures and their occurrence appears to be related to the metabolic activity of the cell [27]. These structures have been shown in other cell types to allow entry of proteins and other substances to the interior of the mitochondria [33]. Recently, small GTP-binding proteins have been shown to be involved in membrane fusion [13] raising the possibility that 19 kDa may play some role in the forma- tion and function of contact points in adrenal mitochondria which may in turn help to circumvent the barrier to

cholesterol transport provided by the outer mitochondrial membrane.

In order to characterize these two GTP-binding proteins and their functional significance, purification of 45 kDa and 19 kDa is now in progress in this laboratory.

Acknowledgements

We would like to thank Dr. Ghanim Almahbobi, Dr. Shane Brown and Mrs. Mary Leydman for their help and advice. The authors are grateful to the National Institute of Health for support by grant # H D 28961.

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gtp-binding proteins in adrenocortical mitochondria

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