The response of individual polypeptides of the mammalian respiratory chain to thyroid hormone

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 268, No. 1, January, pp. 215-220,1989

The Response of Individual Polypeptides of the Mammalian Respiratory Chain to Thyroid Hormone ANN MUTVEI Department

of Biochemistry,

AND

Am-henius Laboratory,

B. DEAN NELSON’ University

of Stockholm, S-106 91 Stockholm, Sweden

Received May 19,1988, and in revised form August 16,1988

The effects of thyroid hormone on the accumulation of inner membrane polypeptides in rat liver mitochondria have been investigated using Western blot analysis. Respiration and mitochondrial protein synthesis were also measured. Levels of the subunits of cytochrome oxidase, the cytochrome beI complex, and the P-subunit of F,-ATPase increase relatively late, requiring 3-6 days of treatment and high doses of hormone. In contrast, respiration increases under conditions in which no significant accumulation of individual subunits is observed. Our results indicate that increased oxidative capacity of mitochondria can be divided into an early response which probably involves metabolic regulation of mitochondrial respiration by hormone and a later response which is due to elevated mitochondrial protein synthesis and the accumulation of polypeptides of the respiratory chain. 0 1989 Academic Press, Inc.

Thyroid hormone regulates the biogenesis of mammalian mitochondria. Injection of thyroid hormone into hypothyroid rats increases the rate of respiration (l-6), the mitochondrial content of heme aa (7), the specific activity of certain enzymes (8, 9), and the rate of mitochondrial protein synthesis (3, 10-13). It is generally assumed that these hormone-induced changes reflect an increase in inner membrane resulting from elevated mitochondrial protein synthesis. However, no studies have been done to show that thyroid hormone actually increases the steady-state content of the polypeptides which participate in respiration. This is of particular importance since it is now established that respiration can be activated within minutes after injection of triiodothyronine (1, 2), whereas increased mitochondrial protein synthesis appears 1 or 2 days after injection of hormone (10-13). In the present study we used Western blot analysis (14) to measure increases in 1 To whom correspondence

should be addressed.

cytochrome oxidase, the cytochrome be, complex, and the P-subunit of F1-ATPase. Our results show that the accumulation of specific polypeptides in the inner membrane of hypothyroid rats treated with hormone is a slow process and can be separated from early stimulation of respiration. MATERIALS

AND

METHODS

Hypothyroidism was produced in male SpragueDawley rats (120 g body weight) either by hypophysectomy or by addition of Tapazol (Sigma) to the drinking water. The Tapazol-induced hypothyroid state was achieved after 4-5 weeks of treatment, as judged by the absence of body weight gain. Hypothyroid rats were injected with triiodothyronine (Ta)* (Sigma) once a day for 3-6 days as indicated in the text and figures. Three experimental protocols were used in which a total of 60 fig T&00 g body

’ Abbreviations used: T3, triiodothyronine; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GPDH, glycerophosphate dehydrogenase.

215

0003-9861/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

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subunits synthesized in the mitochondria (subunit II) or cytosol (subunit VI), and with an antiserum against holo-complex III which recognizes four different subunits of the complex. By contrast, rats injected with the same total amount of hormone (60 pg/lOO g body weight) over a longer period of time (6 days) show significant increases in specific inner membrane polypeptides. This is demonstrated in Fig. 2 for cytochrome oxidase subunits I and II. Although an increase in subunit VI is not apparent in the figure, densitometric analysis and radioactive measurements of bands cut from the nitrocellulose indicated an increase in subunit VI of between l.l- and 1.6-fold. Subunits I and II increased between 1.7- and 1.9-fold, respectively, as shown by both methods of measurement. The time dependency suggests that the accumulation of inner membrane polypeptides is a relatively slow event. Figure 3 shows that raising the dose of T3 by fourfold (40 pg/lOO g body weight/ day for 6 days) results in a general increase in all of the polypeptides of cytochrome oxidase (Figs. 3A and 3B), the cytochrome beI complex (Fig. 3C), and the Fi-ATPase (Fig. 3D) which were tested. The activities of cytochrome oxidase and NADH oxidase in these membranes were also increased from 0.13 -t 0.01 to 0.30 +- 0.02 and from 0.13 RESULTS + 0.02 to 0.36 t- 0.07 pmol/min/mg, respectively. This result shows that the inIn agreement with previous studies from crease in polypeptide content of the memthis laboratory (3) and others (l-6,8,9), in brane is associated with the assembly of zrivo treatment with T3 stimulates respiration by isolated mitochondria. This is functional respiratory chain units. No sigshown for rats injected with a total of 60 pug nificant increases were observed in cytochrome oxidase or NADH oxidase activiTJlOO g body weight administered either from rats injected over 3 days (20 pg/lOO g/day) or 6 days (10 ties in mitochondria with lower doses of T3 or for shorter peripg/lOO g/day) (Table I). ods of time (not shown). Western blot analysis of inner mitoHowever, mitochondrial a-glycerophoschondrial membranes from rats injected phate dehydrogenase was measured to with 20 pg/lOO g/day for 3 days show, howcontrol the response of hypothyroid rats to ever, no increase in the individual peptides hormone. a-GPDH activity was elevated of the cytochrome bc, complex or cytothreefold in rats treated with 20 pg T,/day chrome oxidase (Fig. l), even though mitofor 3 days and fivefold in rats treated with chondrial protein synthesis is increased 10 pg T,/day for 6 days (data not shown). under these conditions (Table I; Refs. (2,3, dehydrogenase 6,10-13). This was demonstrated using an Thus, a-glycerophosphate responds earlier than the other inner antiserum recognizing cytochrome oxidase

weight was injected over a period of 3 days (20 pg/ day/100 g) or 6 days (10 rg/day/lOO g), or in which a total of 240 pg T&00 g body weight was were injected over 6 days (40 pg T,/day/lOO g). Control hypothyroid rats were injected with an equal volume of solvent (0.01 M NaOH) lacking TQ. Liver mitochondria were isolated as described (15). Mitochondrial protein synthesis was measured in vitro by the incorporation of [35S]methionine (3). Oxidative phophorylation at 30°C was measured polarographically in a medium containing 50 mM Tris-Cl, pH 7.5, 100 mM KCl, 5 mM MgCla, 4.5 mM KP, and 10 mM succinate. State 3 respiration was initiated by addition of 0.3 mM ADP. Electrophoresis was carried out on 12.5% SDS polyacrylamide gels in the buffer system of Laemmeli (16). The polypeptides were transferred to nitrocellulose sheets which were subsequently fixed with 0.5% glutaraldehyde (17), blocked with 5% BSA in phosphate-buffered saline (PBS), and incubated overnight with antibodies diluted in 5% BSA/PBS (14). The nitrocellulose was washed three times in PBS containing 0.1% Tween (18). Bound antibodies were detected with ‘%I-protein A (1 X 106cpm/ml) followed by autoradiography. Specific polypeptides were quantified either by cutting the radioactive bands from the nitrocellulose sheets and counting them in a gamma counter or by scanning the autoradiographs with a LKB laser gel scanner. Comparisons between hypothyroid rats and hormonertreated rats were made only when the counts or the densitometric tracings increased linearly with protein. Protein concentrations were measured by the biuret method.

THYROID

HORMONE

AND

MITOCHONDRIAL

TABLE

217

BIOGENESIS

I

THE EFFECT OF THYROID HORMONE ON RESPIRATION AND PROTEIN SYNTHESIS IN MITOCHONDRIA ISOLATED FROM HYPOTHYROID RATS (H) AND HORMONE-INJECTED RATS (T3) Experiment

H

T3

Experiment A (20 fig T,/day for 3 days) Respiration State 4 State 3 Protein synthesis

16.6 f 1.7 84.9 2 2.3 75,000 3~6200

39.1 * 1.4 180.2 k 4.1 129,000 +- 17,200

2.3 2.1 1.7

Experiment B (10 fig T,/day for 6 days) Respiration State 4 State 3 Protein synthesis

16.9 + 2.7 70.4 * 7.9 22,500

37.0 + 0.1 153.5 f 6.5 88,700

2.2 2.2 3.9

Experiment C (40 pg T,/day for 6 days) Respiration State 4 State 3

29.2 + 2.4 96.1 2 4.1

46.3 3~13.7 145.6 f 10.6

1.6 1.5

Note. Rats in Experiment A were injected Experiment B with 60 Fg T&00 g body weight 100 g body weight (40 pg per day for 6 days). protein and the rates of protein synthesis are

T,/H

with 60 pg T&O0 g body weight (20 +g per day for 3 days), in (10 pg per day for 6 days), and in Experiment C with 240 +g Ta/ Respiration rates are given as ng atoms 0 consumed/min/mg cpm/mg protein/l5 min.

membrane proteins measured, suggesting that its expression is regulated differently from the respiratory chain complexes. This is consistent with the results of others showing that a-GPDH is induced (19, 20) under conditions in which no changes were observed in the polypeptides of Complex I (20). The above results show that accumulation of individual inner membrane polypeptides after hormone treatment is a relatively slow process which occurs long after initial increases in respiration rates. DISCUSSION

The steady-state levels of selected polypeptides of the mitochondrial inner membrane were measured in thyroid hormonetreated rats by Western blotting. Antibodies used in. the analysis were directed against polypeptides translated on either mitochondrial or cytoplasmic ribosomes. The results show that although the respi-

ration rate increased twofold after certain hormone injection regimes, there was no increase in the steady-state levels of the individual mitochondrial polypeptides. A general increase in the inner membrane content of both mitochondrially and cytosolically translated polypeptides required relatively long periods of time and/or high doses of hormone. Mitochondrial protein synthesis is activated within 24 h after hormone treatment (lo-13), but does not result in an early accumulation of the individual membrane proteins (Fig. 1). The reason for this delay in the net accumulation of inner membrane polypeptides is not known. It could reflect a relatively slow assembly process in slow growing liver cells. Slow assembly of rat liver cytochrome oxidase has been demonstrated (21), and in this case appears to be limited by insertion of subunit 1 (21, 22). However, slow accumulation of

218 cyiochrome oxidase

MUTVEI

H

T,

complex

T3

III

H

T3

H

AND

T3

II--)

VI--)

FeS -

100

100 50

gg

50

100

Ia

FIG. 1. Quantification of subunits of cytochrome oxidase and the cytochrome bei complex in liver mitochondria from rats given low doses of thyroid hormone for 3 days. Hypothyroid rats (H) were injected with 20 pg T,/lOO g body weight/day for 3 days (total of 60 pg T&00 g body weight). Mitochondrial isolation, electrophoresis, and Western blotting were conducted as described under Materials and Methods. Western blottings were done with antibodies raised against rat liver holo-eytochrome bc, complex (right) and holo-cytochrome oxidase (left). The latter antibody reacted with subunits II and VI of cytochrome oxidase while the former antibody reacted with core proteins I and II, cytochrome ci, and the iron sulfur protein (FeS) of the cytochrome bq complex. Fifty or one hundred micrograms of mitochondrial protein was separated by electrophoresis.

inner membrane polypeptides could also reflect the lack of cytosolic proteins needed for assembly with the mitochondrial translation products. Although rapidly growing tumor cells maintain a large cytosolic pool of unassembled mitochondrial precursor polypeptides (23), it has not been established that similar pools exist in the hepatocyte. In this regard, however, it is of interest that thyroid hormone does not appear to induce a general expansion of the cytosolic pools of mitochondrial precursors (3,13,24). Taylor and Ragan (20) observed no increase in the levels of Complex I subunits in rat liver mitochondria from euthyroid rats injected with thyroid hormone. The absence of a response under these conditions is not surprising in view of the small changes (two- to threefold) which we found for the transition from the hypo- to hyperthyroid states. However, in agreement with the present findings, the same laboratory observed an increase in a-glycerophosphate dehydrogenase activity (20) under conditions which produced no alter-

NELSON

ations in Complex I. Thus, it would appear that a-GPDH and the nuclear encoded subunits of the inner membrane respiratory chain complexes are regulated differently. Expression of (Y-GPDH is related to the occupancy of nuclear receptors for T3 (25-27). The recent reports of multiple nuclear T3 receptors (28) might provide an explanation for the differential response observed in ol-GPDH and the respiratory chain components. The mechanism underlying an early enhancement (min) of mitochondrial respiration by thyroid hormone (1, 2) is clearly different from the more stable changes studied here after 3-6 days of treatment. It has been suggested that rapid activation of respiration involves T3 receptors located in the mitochondrial membrane (29-31), and perhaps the activation of the adenine nucleotide translocator protein (32-34) (however, the importance of mitochondrial receptors has recently been questioned (35)). Short-term increases in respiration are not prevented by inhibitors of protein syn-

cytochrome oxidase I-

80

40

cytochrome

20 IO

5

H

5

1020

80 402010

5

fig

T,

4080

80402010

5

pg

FIG. 2. Quantification of subunits of cytochrome oxidase and the cytochrome be, complex in liver mitochondria from rats given low doses of thyroid hormone for 6 days. Hypophysectomized rats (H) were injected with 10 pg T&00 g body weight/day for 6 days (total of 60 pg T,/lOO g body weight). Increasing amounts of protein were separated. Western blotting was done with antibodies against rat liver cytochrome oxidase subunit I (top) and subunit II (bottom). The latter antiserum also reacts with subunit VI of cytochrome oxidase.

THYROID HORMONE AND MITOCHONDRIAL

BIOGENESIS

219

tion of respiration which is either controlled directly by hormone or via hormone-responsive metabolic signals, and a late activation which requires stimulation of mitochondrial protein synthesis and the accumulation of intact inner membrane lipoprotein complexes.

n cytochrome oxidase

B cyiochrome oxIda%?

ACKNOWLEDGMENTS Antibodies against the P-subunit and the Fi-ATPase were kindly supplied by T. Hundal. This study was supported by funds from the Swedish Natural Science Research Council.

II

5 10 20 40 80

80

40 20

10

5

pg

REFERENCES

c H

T3

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20

40

80

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20 10 5

1-19

B H

T3

Nature

ATPase R----C

10

2040

80

80402010

pg

FIG. 3. Quantification of subunits of cytochrome oxidase, the cytochrome bcl complex and the fl-subunit of F,-ATPase in liver mitochondria from rats given high doses of thyroid hormone for 6 days. Hypothyroid rats (H) were injected with 40 pg T&00 g body weight/day for 6 days (total of 240 Fg T&00 g body weight). Western blotting was done with antibodies against rat cytochrome oxidase subunits I (A) and II (B), holocytochrome bcl complex (C), and the P-subunit of beef Fi-ATPase (D).

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